Detection methods employing hcv core lipid and dna binding domain monoclonal antibodies

ABSTRACT

The present disclosure provides detection methods employing HCV core lipid binding domain and DNA binding domain monoclonal antibodies or antibody fragments. In certain embodiments, the lipid binding domain monoclonal antibody or antibody fragment recognizes an epitope in amino acids 141 to 161 of HCV core protein and the DNA binding domain antibody or antibody fragment recognizes an epitope in amino acids 95-123 (e.g., in amino acids 99-117) of HCV core protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of copending U.S. applicationSer. No. 15/189,646, filed Jun. 22, 2016, which is acontinuation-in-part of U.S. application Ser. No. 15/079,013, filed Mar.23, 2016, which is a divisional of U.S. application Ser. No. 14/138,991,filed Dec. 23, 2013, now U.S. Pat. No. 9,371,374, issued Jun. 21, 2016,which claims priority to U.S. Provisional Patent Application No.61/783,529, filed Mar. 14, 2013, each of which are herein incorporatedby reference in their entireties.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

The subject matter disclosed and claimed herein was made by or on behalfof the parties to a joint research agreement, Icahn School of Medicineat Mount Sinai and Abbott Laboratories, within the meaning of 35 U.S.C.§ 100(h) and § 1.9(e). The joint research agreement was in effect on orbefore the date the claimed invention was made, and the claimedinvention was made as a result of activities undertaken within the scopeof the joint research agreement.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 245,923 Byte ASCII (Text) file named“34340-407_ST25.txt,” created on Aug. 13, 2019.

FIELD OF THE INVENTION

The present disclosure provides detection methods employing HCV corelipid binding domain and DNA binding domain monoclonal antibodies orantibody fragments. In certain embodiments, the lipid binding domainmonoclonal antibody or antibody fragment recognizes an epitope in aminoacids 141 to 161 of HCV core protein and the DNA binding domain antibodyor antibody fragment recognizes an epitope in amino acids 95-123 (e.g.,in amino acids 99-117) of HCV core protein.

BACKGROUND OF THE INVENTION

According to WHO statistics, as many as 170 million people worldwide areinfected by hepatitis C virus (HCV), a viral infection of the liver. 75to 85% of persons infected with HCV progress to chronic infection,approximately 20% of these cases develop complications of chronichepatitis C, including cirrhosis of the liver or hepatocellularcarcinoma after 20 years of infection. The current recommended treatmentfor HCV infections is a combination of interferon and ribavirin drugs,however the treatment is not effective in all cases and the livertransplantation is indicated in hepatitis C-related end-stage liverdisease. At present, there is no vaccine available to prevent HCVinfection, therefore all precautions to avoid infection must be taken.

Thus, patient care, as well as the prevention of transmission ofHepatitis C Virus (HCV) by blood and blood products or by close personalcontact requires extreme vigilance using sensitive detection assays.This creates a need for specific methods for screening and identifyingcarriers of HCV and HCV-contaminated blood or blood products.Serological determination of HCV exposure relies on the detection of HCVpresent in human blood plasma or sera. This can be accomplished bydetection of distinct structural and non-structural proteins encoded bythe virus.

The HCV virus is a (+) sense single-stranded enveloped RNA virus in theHepacivirus genus of the Flaviviridae family. The viral genome isapproximately 10 kb in length and encodes a 3011 amino acid polyproteinprecursor. The HCV genome has a large single open reading frame (ORF)coding for a unique polyprotein. This polyprotein is co- andpost-translationally processed by cellular and viral proteases intothree structural proteins, i.e., core, E1 and E2 and at least sixnon-structural NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins. (Choo etal., Science 244: 359-362 (1989)).

Following HCV exposure, the virus enters a susceptible hepatocyte andviral replication occurs. During an eclipse phase period ofapproximately 10 days, viral presence is not evident (i.e., viral RNAcannot be detected), serum transaminase levels are within normal limits,and no evidence exists of an immune response to HCV (Busch et al.,Transfusion 40:143 (2000)). Typically, about 10 days following exposure,HCV RNA can be detected, often with viral loads between100,000-120,000,000 HCV RNA copies per ml of serum. Typically severalweeks later, an increase in ALT levels is observed, indicatinginflammation of the liver; antibodies are detected an average of about70 days after exposure.

Screening of blood for exposure to HCV, either by the detection ofantibodies to HCV or by the detection of viral-specific molecules (e.g.,HCV RNA or HCV core proteins) in serum/plasma is an integral andimportant part of patient care. Blood or blood products derived fromindividuals identified as having been exposed to HCV, by these tests,are removed from the blood supply and are not utilized for distributionto recipients of blood products (see, e.g., U.S. Pat. No. 6,172,189).These tests may also be utilized in the clinical setting to diagnoseliver disease attributable to HCV infection.

Serologic antibody tests rely on the use of recombinant antigens orsynthetic peptides, representing selected fragments of the viralpolyprotein. The first generation anti-HCV screening tests were based ondetection of antibodies directed against a recombinant protein (HCVgenotype 1a) originating from sequences located in the nonstructuralNS-4 protein (C100-3) (Choo et al., Science 244:359 (1989); Kuo et al.,Science 244:362 (1989)). The first generation assays failed to detectantibodies in approximately 10% of individuals having chronic HCVinfection and up to 10-30% of individuals presenting with acute HCVinfection. The second generation anti-HCV assays have incorporatedrecombinant proteins from three different regions of the HCV genome (HCVgenotype 1a), including amino acid sequences from the core, NS3, and NS4protein (Mimms et al., Lancet 336:1590 (1990); Bresters et al., Vox Sang62:213 (1992)), allowing a marked improvement over the first generationtests in identifying HCV infected blood donors (Aach et al., N Engl JMed 325:1325 (1991); Kleinman et al., Transfusion 32:805 (1992). Thesecond-generation assays detect antibodies in close to 100% of chronicHCV cases (Hino K., Intervirology 37:77 (1994)) and in nearly 100% ofthe acute cases by 12 weeks post infection (Alter et al., N Engl J Med327:1899 (1992); Bresters et al., Vox Sang 62:213 (1992)). The thirdgeneration test includes a recombinant protein expressing amino acidsequences from the NS5 region, as well as antigens from the core, NS3and NS4. Some studies have indicated a slight improvement in sensitivityin comparing the third generation tests to second generation tests (Leeet al., Transfusion 35:845 (1995); Courouce et al. Transfusion34:790-795 (1994)), but this improvement is largely attributed tochanges in the NS3 protein rather than the inclusion of NS5 (Courouce etal., Lancet 343:853 (1994)).

In general, the second and third generation HCV antibody tests detectexposure to HCV about 70 days after exposure. Since HCV establishespersistent, and in many cases lifelong infection, the detection ofantibodies to HCV represents a very efficient method for determiningexposure to HCV. However, antibody testing alone will frequently fail todetect HCV infected individuals during the first 70 days after exposure.

It has been suggested that testing for HCV antigen detects exposure toHCV significantly earlier than antibody testing and represents analternative to nucleic acid testing for detecting exposure to HCV duringthe pre-seroconversion period. The HCV antigen detection test is rapid,simple, may-not require sample extraction or other pretreatment, and isnot as prone to handling errors (e.g., contamination) as may occur inthe HCV RNA tests. Thus, HCV core antigen tests present a practicalalternative to HCV RNA for screening blood donors or for monitoringantiviral therapy.

Existing HCV antigen tests rely on detecting the presence of the HCVcore antigen in serum or plasma. HCV core protein is a structuralprotein of HCV comprising the first 191 amino acids of the polyproteinand that forms the internal viral coat encapsidating the genomic RNA.Two different types of serologic assays have been developed which permitdetection of HCV core antigens in serum. One assay format detects HCVcore antigens in subjects prior to seroconversion and is utilized inscreening blood donors, while the other assay format detects coreantigens only in hepatitis C patients, regardless of their HCV antibodystatus, and is utilized in clinical laboratories to diagnose exposure toHCV or to monitor antiviral therapy. The currently available coreantigen detection assays all use antibodies against the DNA bindingdomain of HCV core which is located at amino acids 1-125 of the coreprotein. The core protein also contains a lipid binding domain that islocated between amino acids 134-171. To date there have been no antigensdescribed from that section of core protein and until now it has beenassumed that core detection required antibodies against the DNA bindingdomain.

Thus, binding proteins that can readily detect HCV core antigen willmarkedly improve the available methods of detection of HCV exposure in apatient. Thus, there is a recognized need for new antibodies that canreadily be employed in screening tests.

SUMMARY OF THE INVENTION

The present disclosure provides detection methods employing HCV corelipid binding domain and DNA binding domain monoclonal antibodies orantibody fragments. In certain embodiments, the lipid binding domainmonoclonal antibody or antibody fragment recognizes an epitope in aminoacids 141 to 161 of HCV core protein and the DNA binding domain antibodyor antibody fragment recognizes an epitope in amino acids 95-123 (e.g.,in amino acids 99-117) of HCV core protein. The DNA binding domain, mayalso be referred to as the RNA binding domain. Both DNA and RNA bindingdomain refer to amino acids 1-125 of the HCV core protein.

In some embodiments, provided herein are methods for the detection ofHCV protein in a test sample comprising: (i) contacting a test samplesuspected of containing HCV with a first antibody, or antigen-bindingportion thereof, directed against an epitope in the DNA (or RNA) bindingdomain of an HCV core protein to form a complex between the firstantibody, or the antigen-binding portion thereof, and the HCV coreprotein located within the test sample, wherein the epitope in the DNAbinding domain is within amino acids 95-123 of full-length HCV coreprotein; (ii) contacting the complex formed in step (i) with a secondantibody, or antigen-binding portion thereof, directed against anepitope in the lipid binding domain of HCV core protein, to form acomplex between the second antibody, or the antigen-binding portionthereof, and the complex formed in step (i), wherein the epitope in thelipid binding domain is within amino acids 141-161 of full-length HCVcore protein; and (iii) detecting the complex formed in step (ii).

In some embodiments, provided herein are methods for the detection ofHCV protein in a test sample comprising: (i) contacting a test samplesuspected of containing HCV with a first antibody, or antigen-bindingportion thereof, directed against an epitope in the lipid binding domainof an HCV core protein, to form a complex between the first antibody, orthe antigen-binding portion thereof, and the HCV core protein locatedwithin the test sample; wherein the epitope in the lipid binding domainis within amino acids 141-161 of full-length HCV core protein, (ii)contacting the complex formed in step (i) with a second antibody, orantigen-binding portion thereof, directed against an epitope in the DNA(or RNA) binding domain of HCV core protein to form a complex betweenthe second antibody, or the antigen-binding portion thereof, and thecomplex formed in step (i), wherein the epitope in the DNA bindingdomain is within amino acids 95-123 of full-length HCV core protein; and(iii) detecting the complex formed in step (ii).

In other embodiments, provide herein are systems or compositionscomprising: (i) a first antibody, or antigen-binding portion thereof,directed against an epitope in the DNA binding domain of HCV coreantigen, wherein the epitope in the DNA binding domain is within aminoacids 95-123 of full-length HCV core protein; and (ii) a secondantibody, or antigen-binding portion thereof, directed against anepitope in the lipid binding domain of HCV core antigen, wherein theepitope in the lipid binding domain is within amino acids 141-161 offull-length HCV core antigen. In certain embodiments, the first andsecond antibodies, or fragments thereof, are in separate or the samecontainer. In further embodiments, the systems further comprise a samplefrom a subject suspect of being infected with HCV.

In other embodiments, the first and/or second antibody, or theantigen-binding portion thereof, is detectably labeled with a label, andwherein the detecting the complex in step (iii) comprises detecting thelabel. In some embodiments, provide herein the HCV core protein in thecomplex is an HCV mini-core protein. In particular embodiments, the HCVcore protein in the complex is a full-length HCV core protein. Inadditional embodiments, the epitope in the DNA binding domain is withinamino acids 95-117 of full-length HCV core protein. In furtherembodiments, the epitope in the DNA binding domain is within amino acids103-109 or 103-110 of full-length HCV core protein. In certainembodiments, the epitope in the DNA binding domain is within amino acids99-123 of full-length HCV core protein. In some embodiments, the epitopein the DNA binding domain is within amino acids 109-113 of full-lengthHCV core protein.

In certain embodiments, the present invention provides a monoclonalantibody that is specifically immunoreactive with the lipid bindingdomain of HCV core antigen. More particularly, the HCV core antigen isamino acid residues 134-171 of HCV. In more particular embodiments, theantibody specifically binds at least one epitope formed by amino acidsequence MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:578). In morespecific embodiments, the antibody is immunoreactive with an epitopeformed by amino acids 141-161, 134-154 and 151-171 of HCV core antigen.

Another aspect of the invention provides a monoclonal antibody that isspecifically immunoreactive with the lipid binding domain of HCV coreantigen, wherein said monoclonal antibody has a heavy chain variabledomain selected from the group consisting of the antibodies listed inFIG. 1A, and a light chain variable domain selected from the groupconsisting of the antibodies listed in FIG. 1B.

It is contemplated that any of the antibodies described herein may beprepared as immunoassay reagents, more particularly, such reagentspreferably are labeled with a detectable label.

In still other embodiments, immunoassay reagents of the inventioncomprise one or more of the antibodies disclosed herein being bound to asolid phase.

The immunoassay reagents comprising the antibodies of the invention mayfurther comprise an additional antibody against an HCV antigen. Forexample, such an additional antibody is an additional anti-coreantibody.

A further aspect of the invention is directed to an immunoassay for thedetection of HCV in a test sample, said immunoassay comprising:

(i) contacting a test sample suspected of containing HCV with a firstantibody directed against HCV core antigen to form a complex betweensaid first antibody and antigen located within said test sample;(ii) contacting said complex formed in step (i) with an antibody to acore lipid binding domain, to form a complex between said antibody to acore lipid binding domain and antigen in the complex formed in step (i)wherein said antibody to a core lipid binding domain is detectablylabeled; and(iii) detecting the label of the complex formed in step (ii).

In more specific embodiments, the immunoassay may further becharacterized in that the first antibody is directed to the DNA bindingdomain of HCV core antigen. In more particular embodiments the antibodyemployed in step (ii) is labeled with a fluorescent label. In exemplaryembodiments, the label is acridinium.

In some embodiments, the immunoassay is one in which the antibody ofstep (i) is coated on solid phase. In specific preferred embodiments,the antibody of step (i) comprises an antibody that is distinct from theantibody of step (ii). Alternatively, the immunoassay is one in whichthe antibody of step (i) comprises an antibody that is the same as theantibody of step (ii).

Any of the immunoassays of the invention may be used on a test sampleobtained from a patient and the method further comprises diagnosing,prognosticating, or assessing the efficacy of a therapeutic/prophylactictreatment of the patient, wherein, if the method further comprisesassessing the efficacy of a therapeutic/prophylactic treatment of thepatient, the method optionally further comprises modifying thetherapeutic/prophylactic treatment of the patient as needed to improveefficacy.

As will be described in further detail herein, it will be understood bythose skilled in the art that any of the immunoassays of the inventionmay be readily adapted for use in an automated system or asemi-automated system.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows the heavy chain variable domains of preferred antibodiesof the present invention. FIG. 1A discloses SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, and 100, respectively, in order ofappearance.

FIG. 1B shows the light chain variable domains of preferred antibodiesof the present invention. FIG. 1B discloses SEQ ID NOS: 302, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 324, 324, 326, 328, 330, 332,334, 336, 338, 340, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362, 364, 366, 368, 370, 370, 370, 372, 374, 376, 376, 378, 380,382, 384, 386, 388, 388, 388, respectively, in order of appearance.

FIGS. 2A and 2B show two representations of a clustering diagram derivedfrom the alignments of FIG. 1A.

FIG. 3 shows the core proteins encoded by HCV, including the precursorp23, mature core p21, and N-terminally deleted 70 and 91 mini-cores.Both p21 mature core and minicore proteins contain the hydrophobicdomain D2 which interacts with lipids. Minicores contain only part ofdomain D1, the RNA-binding domain (aka, the DNA binding domain). Thelocation of an immunogen used in Example 14 (peptides 98-110; SEQ IDN0582) with respect to full length HCV core and mini-cores 70 and 91 isshown in this figure.

FIG. 4 shows a flow diagram of an exemplary purification scheme forisolating HCV core and HCV minicores.

FIGS. 5A and 5B show results from Example 14 where it was shown that HCVRNA is enriched in the heparin/Mn⁺² pellet fraction in the four HCVpatients (P1-P4), but not in two normal controls (N1-N2).

FIG. 6 shows results from Example 14, where minicores are detected bywestern blot in four HCV patients (lanes P1-P4) and not in two normalcontrols (lanes N1, N2). Cell culture supernatant of infected cellsserves as a positive control (lane C).

FIG. 7 shows results of Example 14, where duplicate Western blots ofsample P4 were probed with antibodies directed either to the C-term orN-term portion of the core protein confirms the identity of mini-cores.

FIG. 8A shows the amino acid sequence of Neo4 antibody light chainvariable region. This figure shows the light chain CDRs underlined,which include CDRL1 (RASKSVNEYGYTYMH; SEQ ID NO:585), CDRL2 (LASNLDS;SEQ ID NO:586), and CDRL3 (QHSRELPYT, SEQ ID NO:587).

FIG. 8B shows the amino acid sequence of Neo4 antibody heavy chainvariable region. This figure shows the heavy chain CDRs underlined,which include CDRH1 (GFSITSSVYCWQ; SEQ ID NO:588), CDRH2(RICYDGSVDYSPSITS; SEQ ID NO:589), and CDRH3 (ENHIDYYDTTYPSFDV; SEQ IDNO:590).

FIG. 9A shows the nucleic acid sequence encoding the light chainvariable region of Neo4 (SEQ ID NO:591).

FIG. 9B shows the nucleic acid sequence encoding the heavy chainvariable region of Neo4 (SEQ ID NO:592).

FIG. 10 shows results from Example 18, which demonstrated that whenantibodies Neo4 and 208B-750 are used in combination, the signalintensity for core and 91-minicore are enhanced as compared to when theantibodies are used individually.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides detection methods employing HCV corelipid binding domain and DNA binding domain monoclonal antibodies orantibody fragments. In certain embodiments, the lipid binding domainmonoclonal antibody or antibody fragment recognizes an epitope in aminoacids 141 to 161 of HCV core protein and the DNA binding domain antibodyor antibody fragment recognizes an epitope in amino acids 95-123 (e.g.,in amino acids 99-117) of HCV core protein.

In some embodiments, the present invention describes the development ofmonoclonal antibodies directed against the Hepatitis C Virus coreantigen, specifically, the lipid binding domain of core antigen betweenamino acids 134-171. The immunogen used was a synthetic peptide andscreening of hybridomas utilized both the immunogen peptide and a set ofthree overlapping smaller peptides within the 134-171 region. Inaddition, a recombinant core antigen representing amino acids 1-169 wasused for screening to determine the efficacy of the identifiedmonoclonal antibodies as reactive with HCV core protein. The antibodiesare delineated by their reactivity to the antigen, the immunogenpeptide, and smaller overlapping peptides comprising the immunogen.Binding kinetics of the antibodies to the immunogen peptide weredetermined by SPR (surface plasmon resonance) using a BIAcore 4000instrument. Immunoreactivity to the recombinant core antigen wasdetermined by standard ELISA.

In addition, to show that the monoclonal antibodies of the presentinvention were useful in analyzing the presence of core antigen, coreantigen capture microtiter plate assays were performed by usingmonoclonal antibodies directed to epitopes within the DNA binding domainof HCV core (e.g. amino acids 1-125) as the capture reagent and the134-171-directed antibodies of the present invention as detectionreagents. These assays produced results to demonstrate the utility ofthe 134-171-directed antibodies of the invention for HCV core antigendetection immunoassays. This is the first demonstration of an antigencapture assay that independently targets two major domains of HCV corefor capture and detection. Previously reported core antigen detectionassays use antibodies that bind to epitopes within the DNA bindingdomain (e.g., amino acids 1-125).

To produce the antibodies of the present invention, mice were immunizedwith a synthetic peptide comprised of HCV core genotype 1 consensussequence from amino acids 134-171 linked to BSA. More specifically, theimmunogen had the sequence of:

(SEQ ID NO: 578) MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG.

In addition, the binding of the monoclonal antibodies to three specificN-terminally biotinylated epitope regions also was characterized and isfurther discussed in the Examples. Specifically, the three overlappingepitopes were derived from the above region and had the sequences of:

HCVc 134-154 (SEQ ID NO: 573) MGYIPLVGAPLGGAARALAHG HCVc 141-161(SEQ ID NO: 574) GAPLGGAARALAHGVRVLEDG HCVc 151-171 (SEQ ID NO: 575)LAHGVRVLEDGVNYATGNLPG

The immunogen was conjugated to BSA to produce the antibodies. In otherembodiments, the immunogen was conjugated to TT and fibrils. The TTsequence is often used to provide a more robust immune response in mice.The sequence of the TT conjugate was:

(SEQ ID NO: 577) Ac-Met-Gly-Tyr-Ile-Pro-Leu-Val-Gly-Ala-Pro-Leu-Gly-Gly-Ala-Ala-Arg-Ala-Leu-Ala-His-Gly-Val-Arg-Val-Leu-Glu-Asp-Gly-Val-Asn-Tyr-Ala-Thr-Gly-Asn-Leu-Pro-Gly-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu-NH2 .

The sequence of the fibrils conjugate was:

(SEQ ID NO: 579) Ac-Met-Gly-Tyr-Ile-Pro-Leu-Val-Gly-Ala-Pro-Leu-Gly-Gly-Ala-Ala-Arg-Ala-Leu-Ala-His-Gly-Val-Arg-Val-Leu-Glu-Asp-Gly-Val-Asn-Tyr-Ala-Thr-Gly-Asn-Leu-Pro-Gly-Gln-Gln-Lys-Phe-Gln-Phe-Gln-Phe-Glu- Gln-Gln-NH2.

B-lymphocytes were fused with a myeloma fusion partner to createhybridomas that were then screened for reactivity against the immunogenpeptide, three overlapping peptides within the immunogen peptidesequence, and a recombinant HCV core antigen. Kinetic profiling using aBiacore 4000 allowed for identification of clusters of antibodieswherein the clusters are defined by their ability to bind to theimmunogen peptide, or shorter peptides overlapping the 134-171 region.Combining these results with immunoreactivity, or lack thereof, for therecombinant antigen as determined by ELISA, allowed further delineationof the antibodies into groups with similar characteristics(specificities).

To briefly summarize the screening results discussed in further detailin the Examples, the greatest immune response was seen in mice immunizedwith the peptide linked to BSA. Additionally, the response from thesemice was predominantly focused on the amino acid 141-161 region,although there was also some response to the amino acid 134-154 and151-171 regions as well. With the HCV peptide linked to TT, an immuneresponse was seen, however, this response was not as strong as with BSA.The response was spread over all 3 epitope regions. On the other hand,mice immunized using the amino acid 134-171 peptide linked to a peptidethat would form fibril networks failed to show a significant immuneresponse. The antibodies of the present invention are described infurther detail in FIGS. 1A and 1B, and in the Examples.

The HCV core antigen used for these studies was expressed in E. coli andpurified in a two-step process using IMAC followed by reverse-phase HPLCbased on previously published methods (Boulant et al., J. Virol. (2005),79(17):11353-11365).

In this manner a significant array of monoclonal antibodies that arespecific for the lipid binding domain of HCV core have been produced.These monoclonal antibodies have utility in development of diagnosticassays for the detection of HCV core antigen in the serum and plasma ofinfected individuals. Prior to the present invention, there has been noreported generation of monoclonal antibodies to the multiple epitopeswithin the core amino acid 134-171 region that have shown bindingactivity to the HCV full length core peptide. The availability of themonoclonal antibodies of the present invention allows for thedevelopment of immunoassays for core antigen detection wherein two majordomains of HCV core antigen are targeted. Previous core antigen assaysdescribed the use of monoclonals directed to epitopes within amino acid1-125 (nucleic acid binding domain). Because the previously describedmonoclonal antibodies were only able to target the nucleic acid bindingdomain of HCV core, they were at best inefficient and often ineffectiveat detection of core protein fragments, break-down products, or smallercore proteins derived by internal translation initiation. The presentinvention for the first time overcomes these inadequacies in theprevious assays by providing specific monoclonal antibodies that can beused as reagents to more efficiently and rapidly detect HCV core presentin a test sample.

More particularly, the antibodies described herein are reagents usefulfor detection of HCV core antigen and are useful reagents to facilitateinvestigation of the life cycle of HCV. As noted above, HCV encodedproteins are expressed in a concerted process in which ribosomes bind tothe internal ribosome entry site (IRES) and initiate translation,leading to synthesis of the viral polyprotein, which is cleaved toproduce the classical HCV proteins, p21 core, E1, E2, p′7, and thenonstructural proteins. None of the viral enzymes, including the viralpolymerase, can be made without the initiation of translation in thecore gene region. Because of this temporal relationship, it is believedthat translation events in this region control the expression all HCVproteins. Hence, a complete understanding of the core gene and its geneproducts is essential to understanding the life cycle of the virus andmay shed light on our understanding of mechanisms of viruspathogenicity. Recently, a new family of conserved viral proteins,referred to as minicores have been described (Eng et al., J Virol. 2009April; 83(7):3104-3114). These proteins are encoded in the same readingframe as the core gene but, are believed to be derived from internaltranslation initiation events rather than post-translational processingof the full-length core protein. One of the minicore proteins describedis termed “91 minicore” named for the presumed initiator codon withincore. It is hypothesized that “134 minicore” also exists, being derivedfrom translation initiation at codon 134 which encodes a methionine inmany HCV isolates. However, reagents are not available that allowdetection of minicores that are, essentially, derived from the lipidbinding domain. Such proteins may play an important role in HCVpersistence.

Since the monoclonal antibodies were raised against a linear, syntheticpeptide derived from HCV core 134-171, it is unknown whether they willbind to the native, complete core antigen or processed forms of HCV corethat exist in infected individuals. However, provided that themonoclonal antibodies of the present invention are able at least to bindone or more epitopes presented by the linear HCV core region from aminoacids 134-171, it is contemplated that such binding will be sufficientto render these monoclonal antibodies significantly useful in HCVdetection assays. Some of the monoclonal antibodies of the presentinvention react with recombinant core antigen while others do not,suggesting that within the core amino acid 134-171 region, there areboth linear and conformational epitopes. Antibodies recognizing eitherlinear or conformational epitopes are very useful tools for the study ofvirus assembly within infected cells and the virus life cycle generally.

Finally, these reagents can also be used in immunoassays where it isdesirable to determine the presence of only the lipid binding domain.Since little is known about circulating levels of minicores in infectedindividuals it is possible that they are present at much higher levelsthan core proteins containing the region of amino acids 1-125. Inproviding antibodies that detect HCV core peptides outside of the regionof amino acids 1-125, the present invention provides HCV core antigendetection assays with much greater sensitivity than those currentlyavailable.

Definitions

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CHL CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as LCVR or VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulinmolecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG 1, IgG2, IgG-3, IgG4, IgA1 and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (Winter, et al. U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fcportion of an antibody mediates several important effector functionse.g., cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fc.gamma.R5 and complement Clq,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. At least one aminoacid residue is replaced in the constant region of the antibody, forexample the Fc region of the antibody, such that effector functions ofthe antibody are altered. The dimerization of two identical heavy chainsof an immunoglobulin is mediated by the dimerization of CH3 domains andis stabilized by the disulfide bonds within the hinge region (Huber etal. Nature; 264: 415-20; Thies et al 1999 J Mol Biol; 293: 67-79.).Mutation of cysteine residues within the hinge regions to prevent heavychain-heavy chain disulfide bonds will destabilize dimerization of CH3domains. Residues responsible for CH3 dimerization have been identified(Dall'Acqua 1998 Biochemistry 37: 9266-73.). Therefore, it is possibleto generate a monovalent half-Ig. Interestingly, these monovalent halfIg molecules have been found in nature for both IgG and IgA subclasses(Seligman 1978 Ann Immunol 129: 855-70; Biewenga et al 1983 Clin ExpImmunol 51: 395-400). The stoichiometry of FcRn: Ig Fc region has beendetermined to be 2:1 (West et al. 2000 Biochemistry 39: 9698-708), andhalf Fc is sufficient for mediating FcRn binding (Kim et al 1994 Eur JImmunol; 24: 542-548.). Mutations to disrupt the dimerization of CH3domain may not have greater adverse effect on its FcRn binding as theresidues important for CH3 dimerization are located on the innerinterface of CH3 b sheet structure, whereas the region responsible forFcRn binding is located on the outside interface of CH2-CH3 domains.However the half Ig molecule may have certain advantage in tissuepenetration due to its smaller size than that of a regular antibody. Atleast one amino acid residue may be replaced in the constant region ofthe binding protein of the present disclosure, for example the Fcregion, such that the dimerization of the heavy chains is disrupted,resulting in half DVD Ig molecules. The anti-inflammatory activity ofIgG is completely dependent on sialylation of the N-linked glycan of theIgG Fc fragment. The precise glycan requirements for anti-inflammatoryactivity has been determined, such that an appropriate IgG1 Fc fragmentcan be created, thereby generating a fully recombinant, sialylated IgG1Fc with greatly enhanced potency (Anthony, R. M., et al. (2008) Science320:373-376).

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′).sub.2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites. (See, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Such antibody binding portions are known in the art. (See, e.g.,Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag.New York. 790 pp. (ISBN 3-540-41354-5)). In addition, single chainantibodies also include “linear antibodies” comprising a pair of tandemFv segments (VH—CH1-VH—CH1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions (Zapata etal., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

The term “multivalent binding protein” is used throughout thisspecification to denote a binding protein comprising two or more antigenbinding sites. In one aspect, the multivalent binding protein isengineered to have three or more antigen binding sites, and is generallynot a naturally occurring antibody. Dual variable domain (DVD) bindingproteins comprise two or more antigen binding sites and are tetravalentor multivalent binding proteins. DVDs as described herein can bemonospecific, i.e., capable of one antigen such as HCV core protein, ormultispecific, i.e. capable of binding two or more antigens. DVD bindingproteins comprising two heavy chain DVD polypeptides and two light chainDVD polypeptides are referred to as DVD-Ig, and are described forexample in U.S. Pat. No. 7,612,181, the disclosure of which is hereinincorporated by reference in its entirety. Each half of a DVD-Igcomprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site.

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

An “immunoglobulin constant domain” refers to a heavy or light chainconstant domain. Human IgG heavy chain and light chain constant domainamino acid sequences are known in the art.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), each mAbis directed against a single determinant on the antigen. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the presentdisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II C, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom H. R. (1997) TIB Tech.15:62-70; Azzazy H., and Highsmith W. E. (2002) Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see, Taylor, L. D., et al.(1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A. and Green L. L.(2002) Current Opinion in Biotechnology 13:593-597; Little M. et al.(2000) Immunology Today 21:364-370) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. Such recombinant human antibodies canbe subjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) such that theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Exemplary affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. BidlTechnology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al, J. Mol.Biol. 226:889-896 (1992) and selective mutation at selective mutagenesispositions, contact or hypermutation positions with an activity enhancingamino acid residue as described in U.S. Pat. No. 6,914,128 B 1.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. Also “humanized antibody” is an antibody or a variant,derivative, analog or fragment thereof which immunospecifically binds toan antigen of interest and which comprises a framework (FR) regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. In one aspect, a humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. In someembodiments, a humanized antibody contains both the light chain as wellas at least the variable domain of a heavy chain. The antibody also mayinclude the CHL hinge, CH2, CH3, and CH4 regions of the heavy chain. Insome embodiments, a humanized antibody only contains a humanized lightchain. In some embodiments, a humanized antibody only contains ahumanized heavy chain. In specific embodiments, a humanized antibodyonly contains a humanized variable domain of a light chain and/orhumanized heavy chain.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the herein systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although certain embodiments useKabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region. As used herein,the term “germline antibody gene” or “gene fragment” refers to animmunoglobulin sequence encoded by non-lymphoid cells that have notundergone the maturation process that leads to genetic rearrangement andmutation for expression of a particular immunoglobulin. (See, e.g.,Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchaloniset al., Adv Exp Med. Biol. 484:13-30 (2001)). One of the advantagesprovided by various embodiments of the present disclosure stems from therecognition that germline antibody genes are more likely than matureantibody genes to conserve essential amino acid sequence structurescharacteristic of individuals in the species, hence less likely to berecognized as from a foreign source when used therapeutically in thatspecies.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, preferably at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR. A humanized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. Preferably, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

As used herein, the term “neutralizing” refers to counteracting thebiological activity of an antigen when a binding protein specificallybinds the antigen. In one aspect, the neutralizing binding protein bindsthe cytokine and reduces its biologically activity by at least about20%, 40%, 60%, 80%, 85% or more.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it recognizes its target antigen in a complexmixture of proteins and/or macromolecules. Antibodies are said to “bindto the same epitope” if the antibodies cross-compete (one prevents thebinding or modulating effect of the other). In addition structuraldefinitions of epitopes (overlapping, similar, identical) areinformative, but functional definitions are often more relevant as theyencompass structural (binding) and functional (modulation, competition)parameters.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore® system(BIAcore International AB, a GE Healthcare company, Uppsala, Sweden andPiscataway, N.J.). For further descriptions, see Jonsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “Kon”, as used herein, is intended to refer to the on rateconstant for association of a binding protein (e.g., an antibody) to theantigen to form the, e.g., antibody/antigen complex as is known in theart. The “Kon” also is known by the terms “association rate constant”,or “ka”, as used interchangeably herein. This value indicating thebinding rate of an antibody to its target antigen or the rate of complexformation between an antibody and antigen.

The term “Koff”, as used herein, is intended to refer to the off rateconstant for dissociation, or “dissociation rate constant”, of a bindingprotein (e.g., an antibody) from the, e.g., antibody/antigen complex asis known in the art. This value indicates the dissociation rate of anantibody from its target antigen or separation of Ab-Ag complex overtime into free antibody and antigen.

The term “KD” as used herein, is intended to refer to the “equilibriumdissociation constant”, and refers to the value obtained in a titrationmeasurement at equilibrium, or by dividing the dissociation rateconstant (koff) by the association rate constant (kon). The associationrate constant, the dissociation rate constant and the equilibriumdissociation constant are used to represent the binding affinity of anantibody to an antigen. Methods for determining association anddissociation rate constants are well known in the art. Usingfluorescence-based techniques offers high sensitivity and the ability toexamine samples in physiological buffers at equilibrium. Otherexperimental approaches and instruments such as a BIAcore® (biomolecularinteraction analysis) assay can be used (e.g., instrument available fromBIAcore International AB, a GE Healthcare company, Uppsala, Sweden).Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available fromSapidyne Instruments (Boise, Id.) can also be used.

“Label” and “detectable label” mean a moiety attached to a specificbinding partner, such as an antibody or an analyte, e.g., to render thereaction between members of a specific binding pair, such as an antibodyand an analyte, detectable, and the specific binding partner, e.g.,antibody or analyte, so labeled is referred to as “detectably labeled.”Thus, the term “labeled binding protein” as used herein, refers to aprotein with a label incorporated that provides for the identificationof the binding protein. In one aspect, the label is a detectable markerthat can produce a signal that is detectable by visual or instrumentalmeans, e.g., incorporation of a radiolabeled amino acid or attachment toa polypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y,99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); chromogens,fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, luciferase, alkalinephosphatase); chemiluminescent markers; biotinyl groups; predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are described herein. In this regard, the moiety itself maynot be detectably labeled but may become detectable upon reaction withyet another moiety. Use of “detectably labeled” is intended to encompassthe latter type of detectable labeling.

The term “conjugate” refers to a binding protein, such as an antibody,chemically linked to a second chemical moiety, such as a therapeutic orcytotoxic agent. The term “agent” is used herein to denote a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. In one aspect, thetherapeutic or cytotoxic agents include, but are not limited to,pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, the conjugate antibody may be a detectably labeledantibody used as the detection antibody.

The terms “isolated polynucleotide” and “isolated nucleotide molecule”as used interchangeably herein mean a polynucleotide (e.g., of genomic,cDNA, or synthetic origin, or some combination thereof) that, is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” or “isolated nucleotide molecule” is found innature, or does not occur in nature as part of a larger sequence. An“isolated polynucleotide” or “isolated nucleotide molecule” may beoperably linked to a polynucleotide that it is not linked to in nature.

The terms “regulate” and “modulate” as used interchangeably herein referto a change or an alteration in the activity of a molecule of interest(e.g., the biological activity of a cytokine). Modulation may be anincrease or a decrease in the magnitude of a certain activity orfunction of the molecule of interest. Exemplary activities and functionsof a molecule include, but are not limited to, binding characteristics,enzymatic activity, cell receptor activation, and signal transduction.Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of a cytokine). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in WO01/83525.

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a mammal, including a primate (for example, a human,a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, a mouse, a whale), a bird (e.g., a duck or agoose), and a shark. Preferably, the patient or subject is a human, suchas a human being treated or assessed for a disease, disorder orcondition, a human at risk for a disease, disorder or condition, a humanhaving a disease, disorder or condition, and/or human being treated fora disease, disorder or condition.

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood), plasma, serum,urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

“Component,” “components,” and “at least one component,” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure, “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negativecontrol”) or to contain analyte (“positive control”). A positive controlcan comprise a known concentration of analyte. “Control,” “positivecontrol,” and “calibrator” may be used interchangeably herein to referto a composition comprising a known concentration of analyte. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) should be generallyapplicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte (e.g.,polypeptide of interest) may entail release of the analyte from anyendogenous binding proteins present in the sample. A pretreatmentreagent may be homogeneous (not requiring a separation step) orheterogeneous (requiring a separation step). With use of a heterogeneouspretreatment reagent there is removal of any precipitated analytebinding proteins from the test sample prior to proceeding to the nextstep of the assay.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen (or fragmentthereof) and an antibody (or antigenically reactive fragment thereof))refer to the selective reactivity of the interaction. The phrase“specifically binds to” and analogous phrases refer to the ability ofantibodies (or antigenically reactive fragments thereof) to bindspecifically to analyte (or a fragment thereof) and not bindspecifically to other entities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes, fragments, and variants (includingfragments of variants) thereof, whether isolated or recombinantlyproduced.

Monoclonal Antibodies

FIGS. 1A and 1B show the sequences of various antibodies that have beendetermined to be specific for HCV core antigen and more particularly,have been determined to be specific for the lipid binding domain of HCVcore antigen. It has been found that these monoclonal antibodies arespecifically immunoreactive with the lipid binding domain of HCV coreantigen. More specifically, it is found that the antibodies of thepresent invention specifically bind at least one epitope formed by aminoacid sequence MGYIPLVGAPLGGAARALAHGVRVLED GVNYATGNLPG (SEQ ID NO:578).More particularly, the monoclonal antibodies at least are immunoreactivewith an epitope formed by amino acids 141-161, 134-154 and 151-171 ofHCV core antigen. Given the disclosure of these monoclonal antibodies,the present invention contemplates the uses thereof in specificimmunoassays to facilitate a rapid and efficient detection of thepresence of HCV in a test sample by determining the presence of HCV coreantigen in such a test sample.

The anti-HCV core binding proteins, including monoclonal antibodies andany derivative (e.g., a fragment or variant) thereof that comprises theCDRs of the heavy and light chains of the monoclonal antibodiesdescribed herein (see FIGS. 1A and 1B) provided that such a derivativeretains the property of binding specifically to HCV core protein lipidbinding domain, can be used in immunoassays for diagnosing or prognosinghepatitis C virus infection in a mammal. As used throughout the presentdisclosure, “mammal” includes humans and non-human primates, as well asother animals. It will be understood that a target analyte in theimmunoassays and related methods is the lipid domain of HCV coreprotein, and hence the target analyte is HCV core protein which would bepresent in the sample, such as for example, after HCV infection.Additionally, it should be understood that the immunoassay may detecttwo or more target analytes provided that at least one of the analytesis HCV core protein, the second or additional target analyte may beanother core protein analyte (e.g., the DNA binding domain of HCV coreprotein) or may be an analyte that is not HCV core protein.

The nucleotide (DNA) sequences and deduced protein sequences encodingthe heavy and light chain variable domains of anti-HCV core monoclonalantibodies were obtained by immunizing mice with a synthetic peptidecomprised of HCV core genotype 1 consensus sequence from amino acids134-171 and a tetanus toxoid (TT) peptide sequence. In some embodiments,the amino acid 134-171 sequence was conjugated to BSA. However, in otherembodiments, the synthetic peptide also was conjugated to the TTsequence as this is often used to provide a more robust immune responsein mice, by methods known to those skilled in the art such as thosedescribed in detail herein below and in, for example, Goding, J. W.1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press,Inc., NY, N.Y., pp. 56 97. Briefly, to produce a human-human hybridoma,a human lymphocyte donor is selected. A donor who is known as infectedwith HCV (where infection has been shown for example by the presence ofanti-virus antibodies in the blood or by virus culture) may serve as asuitable lymphocyte donor. Lymphocytes can be isolated from a peripheralblood sample or spleen cells may be used if the donor is subject tosplenectomy. Epstein-Barr virus (EBV) can be used to immortalize humanlymphocytes or a human fusion partner can be used to produce human-humanhybridomas. Primary in vitro immunization with peptides can also be usedin the generation of human monoclonal antibodies. Antibodies secreted bythe immortalized cells are screened to determine the clones that secreteantibodies of the desired specificity. For monoclonal anti-HCV coreantibodies, the antibodies must bind to HCV core protein and morespecifically, the lipid binding domain of HCV core protein respectively.Cells producing antibodies of the desired specificity are selected.Other methods for obtaining monoclonal antibodies can be used, as knownin the art. The Examples below describes how the anti-HCV coremonoclonal antibodies were obtained and characterized followingisolation of mRNA from hybridoma cells grown in cell culture. Deducedamino acid sequences of the heavy and light chain variable regions forthe anti-HCV core monoclonal antibodies of the present invention arelisted in FIG. 1A and FIG. 1B, respectively.

The deduced amino acid sequences of the heavy and the light chaindomains were assigned SEQ ID NOs and the corresponding cDNAs sequencesencoding the same are shown in the Sequence Table in Appendix A.

The cDNA sequences set forth in the Sequence Table represent exemplaryembodiments of the disclosed cDNAs. Variations are contemplated in thecDNA sequences shown therein. Such variations include those that willresult in a nucleic acid sequence that is capable of directingproduction of analogs of the corresponding protein shown in the SequenceTable. It will be understood that due to the degeneracy of the geneticcode, many substitutions of nucleotides may be made that will lead to aDNA sequence that remains capable of directing production of thecorresponding protein or its analogs. All such variant DNA sequencesthat are functionally equivalent to any of the sequences describedherein, are encompassed by the present disclosure.

A variant of any of the binding proteins (as exemplified by monoclonalantibodies of the invention shown in FIGS. 1A and 1B) described hereinmeans a protein (or polypeptide) that differs from a given protein(e.g., an anti-HCV core monoclonal antibody) in amino acid sequence bythe addition (e.g., insertion), deletion, or conservative substitutionof amino acids, but that retains the biological activity of the givenprotein. A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101, which is incorporated herein by reference). Substitution ofamino acids having similar hydrophilicity values can result in peptidesretaining biological activity, for example immunogenicity, as isunderstood in the art. In one aspect, substitutions are performed withamino acids having hydrophilicity values within ±2 of each other. Boththe hydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. “Variant” also can be used to describe apolypeptide or fragment thereof that has been differentially processed,such as by proteolysis, phosphorylation, or other post-translationalmodification, yet retains its biological activity or antigen reactivity,e.g., the ability to bind to IL-18. Use of “variant” herein is intendedto encompass fragments of a variant unless otherwise contradicted bycontext.

The antibodies of the present invention or antigen binding fragments ofthose antibodies (e.g., fragments that comprise the heavy and lightchain CDRs of the antibodies of the present invention) may also beproduced by genetic engineering. For example, the technology forexpression of both heavy and light chain genes in E. coli is the subjectof the PCT patent applications; publication number WO 901443, W0901443,and WO 9014424 and in Huse et al., 1989 Science 246:1275 1281. Thepresent disclosure also encompasses an isolated recombinant vectorcomprising a nucleic acid molecule as described herein, as well as ahost cell comprising such a recombinant vector. A vector is a nucleicacid molecule, which may be a construct, capable of transporting anothernucleic acid to which it has been linked. A vector may include anypreferred or required operational elements. Preferred vectors are thosefor which the restriction sites have been described and which containthe operational elements needed for transcription of the nucleic acidsequence. Such operational elements include for example at least onesuitable promoter, at least one operator, at least one leader sequence,at least one terminator codon, and any other DNA sequences necessary orpreferred for appropriate transcription and subsequent translation ofthe nucleic acid sequence. Such vectors contain at least one origin ofreplication recognized by the host organism along with at least oneselectable marker and at least one promoter sequence capable ofinitiating transcription of the nucleic acid sequence. A vector may be aplasmid into which additional DNA segments may be ligated. A vector maybe a viral vector, wherein additional DNA segments may be ligated intothe viral genome. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as aplasmid is the most commonly used form of vector. However, the presentdisclosure is intended to include such other forms of expressionvectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

Sequences that are operably linked are in a relationship permitting themto function in their intended manner. A control sequence operably linkedto a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. Operably linked sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. Expression control sequences are polynucleotide sequencesthat are necessary to effect the expression and processing of codingsequences to which they are ligated. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation signals; sequences that stabilize cytoplasmic mRNA;sequences that enhance translation efficiency (i.e., Kozak consensussequence); sequences that enhance protein stability; and when desired,sequences that enhance protein secretion. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. Control sequences include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

A host cell may be transformed with a vector that introduces exogenousDNA into a host cell in order to render that cell one that recombinantlyproduces the antibodies of the present invention. Transformation mayoccur under natural or artificial conditions using various methods wellknown in the art. Transformation may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method is selected based on the host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, lipofection, and particle bombardment.Transformed cells include stably transformed cells in which the insertedDNA is capable of replication either as an autonomously replicatingplasmid or as part of the host chromosome, and cells which transientlyexpress the inserted DNA or RNA for limited periods of time.

Suitable host organisms include for example a eukaryotic cell systemsuch as but not limited to cell lines such as HeLa, MRC-5 or CV-1. Hostorganisms such as host cells are cultured under conditions appropriatefor amplification of the vector and expression of the protein, as wellknown in the art. Expressed recombinant proteins may be detected by anyof a number of methods also well known in the art.

Although the HCV detection aspects of the present invention merely needthe antibodies to be monoclonal antibodies such that they specificallyrecognize HCV core antigen, it may in some embodiments be desirable toproduce humanized versions of the antibodies of the present invention.“Humanized” antibodies and production thereof is well known to those ofskill in the art. General reviews of “humanized” antibodies are providedby Morrison S., 1985 Science 229:1202 and by Oi et al., 1986BioTechniques 4:214. Suitable “humanized” antibodies can bealternatively produced by CDR or CEA substitution (Jones et al., 1986Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedler et al.1988 J. Immunol. 141:4053, the entire disclosures of which areincorporated herein by reference).

In other embodiments, the monoclonal antibodies of the present inventionmay serve as useful starting materials for the production of engineeredand derivatized binding proteins including dual variable domainimmunoglobulin (DVD-Ig) binding proteins comprising one or more anti-HCVmonoclonal antibodies as described herein. For example, DVD-Ig's withunique binding affinities for HCV core protein may be produced, asdescribed for example in U.S. Pat. No. 7,612,181, the entire disclosureof which is hereby incorporated by reference. DVD-Ig binding proteinsare capable of binding one or more targets. Preferably the bindingprotein comprises a polypeptide chain comprising VD1-(X1)n-VD2-C—(X2)n,wherein VD1 is a first variable domain, VD2 is a second variable domain,C is a constant domain, X1 represents an amino acid or polypeptide, X2represents an Fc region and n is 0 or 1. The binding protein can begenerated using various techniques.

In exemplary techniques, the DVD-Ig can be formed with four polypeptidechains which form four functional antigen binding sites. Thus, forexample, the DVD-Ig is capable of binding HCV core protein. The bindingprotein can be capable of modulating a biological function of HCV coreprotein, or of neutralizing HCV core protein. Exemplary such bindingproteins have at least one heavy chain variable domain comprising anamino acid sequence of at least 90% identity with one of the antibodiesof the present invention and at least the corresponding light chainvariable domain comprising an amino acid sequence having at least 90%identity with a sequence of that light chain variable domain.

The variable domains of a DVD binding protein can be obtained fromparent antibodies, including polyclonal and monoclonal antibodiescapable of binding antigens of interest. The monoclonal antibodies thatspecifically bind to HCV core protein described herein are suitableparent antibodies. Generally, antibodies used for the DVD bindingprotein may be naturally occurring or may be generated by recombinanttechnology.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including those asdescribed herein for preparing the anti-HCV core protein monoclonalantibodies, and those known in the art and taught, for example, inHarlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Hammerling, et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (saidreferences incorporated by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Hybridomas are selected, cloned and further screened fordesirable characteristics, including robust hybridoma growth, highantibody production and desirable antibody characteristics, as discussedin Example 1 below. Hybridomas may be cultured and expanded in vivo insyngeneic animals, in animals that lack an immune system, e.g., nudemice, or in cell culture in vitro. Methods of selecting, cloning andexpanding hybridomas are well known to those of ordinary skill in theart. In a preferred embodiment, the hybridomas are mouse hybridomas. Inanother preferred embodiment, the hybridomas are produced in anon-human, non-mouse species such as rats, sheep, pigs, goats, cattle orhorses. In another embodiment, the hybridomas are human hybridomas, inwhich a human non-secretory myeloma is fused with a human cellexpressing an antibody capable of binding a specific antigen.

Recombinant monoclonal antibodies are also generated from single,isolated lymphocytes using a procedure referred to in the art as theselected lymphocyte antibody method (SLAM), as described in U.S. Pat.No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S. et al.(1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, singlecells secreting antibodies of interest, e.g., lymphocytes derived froman immunized animal, are identified, and, heavy- and light-chainvariable region cDNAs are rescued from the cells by reversetranscriptase-PCR and these variable regions can then be expressed, inthe context of appropriate immunoglobulin constant regions (e.g., humanconstant regions), in mammalian host cells, such as COS or CHO cells.The host cells transfected with the amplified immunoglobulin sequences,derived from in vivo selected lymphocytes, can then undergo furtheranalysis and selection in vitro, for example by panning the transfectedcells to isolate cells expressing antibodies to the antigen of interest.The amplified immunoglobulin sequences further can be manipulated invitro, such as by in vitro affinity maturation methods such as thosedescribed in PCT Publication WO 97/29131 and PCT Publication WO00/56772.

Monoclonal antibodies are also produced by immunizing a non-human animalcomprising some, or all, of the human immunoglobulin locus with anantigen of interest. In a preferred embodiment, the non-human animal isa XENOMOUSE® transgenic mouse, an engineered mouse strain that compriseslarge fragments of the human immunoglobulin loci and is deficient inmouse antibody production. See, e.g., Green et al. Nature Genetics7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615,5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO91/10741, published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994,WO 96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654,published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999,WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24,2000 and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human Mabs. The XENOMOUSE®transgenic mouse contains approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of whichare hereby incorporated by reference.

In vitro methods also can be used to make the parent antibodies, whereinan antibody library is screened to identify an antibody having thedesired binding specificity. Methods for such screening of recombinantantibody libraries are well known in the art and include methodsdescribed in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kanget al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No.WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland etal. PCT Publication No. WO 92/15679; Breitling et al. PCT PublicationNo. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al.,Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734;Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991)Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patentapplication publication 20030186374, and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

Parent antibodies can also be generated using various phage displaymethods known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles that carry thepolynucleotide sequences encoding them. In a particular, such phage canbe utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage expressing an antigen binding domain that binds the antigen ofinterest can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies as described herein include thosedisclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ameset al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al.,Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);Burton et al., Advances in Immunology 57:191-280 (1994); PCT applicationNo. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108; each of which is incorporated hereinby reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of parentantibodies. One type of alternative expression system is one in whichthe recombinant antibody library is expressed as RNA-protein fusions, asdescribed in PCT Publication No. WO 98/31700 by Szostak and Roberts, andin Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, a covalent fusion is created between anmRNA and the peptide or protein that it encodes by in vitro translationof synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic,at their 3′ end. Thus, a specific mRNA can be enriched from a complexmixture of mRNAs (e.g., a combinatorial library) based on the propertiesof the encoded peptide or protein, e.g., antibody, or portion thereof,such as binding of the antibody, or portion thereof, to the dualspecificity antigen. Nucleic acid sequences encoding antibodies, orportions thereof, recovered from screening of such libraries can beexpressed by recombinant means as described above (e.g., in mammalianhost cells) and, moreover, can be subjected to further affinitymaturation by either additional rounds of screening of mRNA-peptidefusions in which mutations have been introduced into the originallyselected sequence(s), or by other methods for affinity maturation invitro of recombinant antibodies, as described above.

In another approach the parent antibodies can also be generated usingyeast display methods known in the art. In yeast display methods,genetic methods are used to tether antibody domains to the yeast cellwall and display them on the surface of yeast. In particular, such yeastcan be utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Examples of yeast display methods that can be used to make the parentantibodies include those disclosed in Wittrup, et al. U.S. Pat. No.6,699,658 incorporated herein by reference.

The monoclonal antibodies described herein can be further modified togenerate CDR grafted and Humanized parent antibodies. CDR-grafted parentantibodies comprise heavy and light chain variable region sequences froma human antibody wherein one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of murine antibodies capable of bindingantigen of interest. A framework sequence from any human antibody mayserve as the template for CDR grafting. However, straight chainreplacement onto such a framework often leads to some loss of bindingaffinity to the antigen. The more homologous a human antibody is to theoriginal murine antibody, the less likely the possibility that combiningthe murine CDRs with the human framework will introduce distortions inthe CDRs that could reduce affinity. Therefore, it is preferable thatthe human variable framework that is chosen to replace the murinevariable framework apart from the CDRs has at least a 65% sequenceidentity with the murine antibody variable region framework. It is morepreferable that the human and murine variable regions apart from theCDRs have at least 70% sequence identify. It is even more preferablethat the human and murine variable regions apart from the CDRs have atleast 75% sequence identity. It is most preferable that the human andmurine variable regions apart from the CDRs have at least 80% sequenceidentity. Methods for producing such antibodies are known in the art(see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.html;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.ukiabout.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pub s/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nliaboutjraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edui;www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;www.ibt.unam.mx/virV_-mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.chLabout.honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.ukfabout.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stataim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Webpages/Pept/spottech.html;wwwjerini.de/fr roducts.htm; www.patents.ibm.con/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

Parent monoclonal antibodies may be selected from various monoclonalantibodies capable of binding specific targets including, or in additionto, HCV proteins, as well known in the art.

Parent monoclonal antibodies may also be selected from varioustherapeutic antibodies approved for use, in clinical trials, or indevelopment for clinical use, particularly those that may be applicablein treating symptoms of HCV infection, or in treating conditions ordiseases that co-exist with HCV infection, such as cancer, includingparticularly hepatocellular carcinoma.

As noted throughout the present invention, it may be desirable to labelthe antibodies of the present invention. A labeled antibody (or abinding protein derived from one of the antibodies of the presentinvention) comprises the antibody, which is derivatized or linked toanother functional molecule (e.g., another peptide or protein). Forexample, the monoclonal antibody can be derivatized by functionallylinking it (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetectable agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate association of the binding proteinwith another molecule (such as a streptavidin core region or apolyhistidine tag).

Useful detectable agents with which monoclonal antibody may bederivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. The antibody may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When derivatized with a detectable enzyme, thedetection is achieved by adding additional reagents that the enzyme usesto produce a detectable reaction product. For example, when thedetectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. A monoclonal antibody of the invention mayalso be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding, or vice versa.

While the compositions of the present invention have demonstrated use indiagnostic applications for determining the presence of HCV core antigenin a test sample, it is contemplated that the compositions of thepresent invention also may serve a diagnostic or therapeutic purpose forin vivo administration to a mammal. Thus, in some embodiments, thepresent invention provides pharmaceutical and diagnostic compositionscomprising one or more anti-HCV core binding proteins disclosed hereinas an active ingredient. Pharmaceutical or diagnostic compositions maycomprise any monoclonal antibody described herein, or any combinationthereof, and a pharmaceutically acceptable carrier, diluent and/orexcipient. Generally, the pharmaceutical and diagnostic compositions areprepared by combining the active ingredient with the carrier, diluentand/or excipient.

The compositions comprising binding proteins as described herein are foruse in, but not limited to, diagnosing, detecting, or monitoring adisorder, but may also find use in preventing, treating, managing, orameliorating of a disorder or one or more symptoms thereof, and/or inresearch. In a specific embodiment, a composition comprises one or moremonoclonal antibodies of the present invention or a binding proteinderived from one or more of the monoclonal antibodies of the presentinvention. In another embodiment, the composition comprises one or moremonoclonal antibodies or binding proteins derived therefrom as describedherein and one or more diagnostic, prophylactic or therapeutic agentsother than monoclonal antibodies or binding proteins derived therefromas described herein.

Immunoassays

Immunoassays according to the present disclosure include such techniquescommonly recognized in the art, including for example radioimmunoassay,Western blot assay, immunofluorescent assay, enzyme immunoassay,chemiluminescent assay, immunohistochemical assay, immunoprecipitationand the like. Standard techniques known in the art for ELISA arewell-known and described for example in Methods in Immunodiagnosis, 2ndEdition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbellet al., Methods of Immunology, W. A. Benjamin, Inc., 1964, both of whichare incorporated herein by reference Immunoassays may be a direct,indirect, competitive, or noncompetitive immunoassay as described in theart (Oellerich, M. 1984. J. Clin. Chem. Clin. BioChem 22:895 904).Biological samples appropriate for such detection assays include, butare not limited to blood, plasma, serum, liver, saliva, lymphocytes orother mononuclear cells.

In preferred embodiments, the antibodies described herein are used inimmunoassays specific for the detection of HCV. Examples include, butare not limited to, sandwich immunoassay, radioisotope detection(radioimmunoassay (MA)) and enzyme detection (enzyme immunoassay (EIA)or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISAassays, R&D Systems, Minneapolis, Minn.)), competitive inhibitionimmunoassay (e.g., forward and reverse), fluorescence polarizationimmunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT),bioluminescence resonance energy transfer (BRET), and homogeneouschemiluminescent assay, etc. In a SELDI-based immunoassay, a capturereagent that specifically binds an analyte of interest such as HCV core(or a fragment thereof) is attached to the surface of a massspectrometry probe, such as a pre-activated protein chip array. Theanalyte (or a fragment thereof) is then specifically captured on thebiochip, and the captured analyte (or a fragment thereof) is detected bymass spectrometry. Alternatively, the analyte (or a fragment thereof)can be eluted from the capture reagent and detected by traditional MALDI(matrix-assisted laser desorption/ionization) or by SELDI. Achemiluminescent microparticle immunoassay, in particular one employingthe ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park,Ill.), is an example of a preferred immunoassay.

An immunoassay for determining the presence or amount of human hepatitisC virus in a sample may comprise, for example, combining an HCV coreprotein binding protein with the sample for a time sufficient for thebinding protein to bind to any human hepatitis C virus that may bepresent in the sample, and determining the presence or amount of humanhepatitis C virus present in the sample based on specific binding of thebinding protein to the human hepatitis C virus core protein. Thedisclosure also encompasses an immunoassay device for detecting thepresence or absence of human HCV in a sample, wherein the devicecomprises any of the antibodies described herein immobilized on a solidsupport. The anti-HCV core antibodies and any analogs thereof may beprepared in the form of a kit, alone, or in combinations with otherreagents such as secondary antibodies, for use in immunoassays.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when an anti-HCV coreantibody of the present invention is employed as an immunodiagnosticreagent and/or in an analyte immunoassay kit. The test sample cancomprise further moieties in addition to the HCV core antigen, includingfor example, antibodies, antigens, haptens, hormones, drugs, enzymes,receptors, proteins, peptides, polypeptides, oligonucleotides and/orpolynucleotides. For example, the sample can be a whole blood sampleobtained from a subject. It can be necessary or desired that a testsample, particularly whole blood, be treated prior to immunoassay asdescribed herein, e.g., with a pretreatment reagent. Even in cases wherepretreatment is not necessary (e.g., most urine samples), pretreatmentoptionally may be performed (e.g., as part of a regimen on a commercialplatform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassay and kits of the present disclosure. The pretreatmentoptionally comprises: (a) one or more solvents (e.g., methanol andethylene glycol) and optionally, salt, (b) one or more solvents andsalt, and optionally, detergent, (c) detergent, or (d) detergent andsalt. Pretreatment reagents are known in the art, and such pretreatmentcan be employed as has been previously described, e.g., as used forassays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers (AbbottLaboratories, Abbott Park, Ill.), as described in the literature (see,e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody Assay Evaluatedfor Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973(1990), and Wallemacq et al., Evaluation of the New AxSYM CyclosporineAssay Comparison with TDx Monoclonal Whole Blood and EMIT CyclosporineAssays, Clin. Chem. 45: 432-435 (1999)), and/or as commerciallyavailable. Additionally, pretreatment can be performed as described inU.S. Pat. No. 5,135,875, European Patent Pub. No. 0 471 293, U.S.Provisional Patent App. 60/878,017, filed Dec. 29, 2006, and U.S. PatentApp. Pub. No. 2008/0020401 (incorporated by reference in its entiretyfor its teachings regarding pretreatment).

With use of a pretreatment reagent the assay is rendered more sensitiveby disruption of preformed/preexisting immune complexes or viralparticles in the test sample. In such a pretreated test sample, theanti-HCV core antibody in the sample is separated from the antigen andthe remaining antigen in the sample is then tested for the presence ofHCV core antigen using the monoclonal antibodies of the presentinvention. The HCV core antigen in the test sample is thus subjected toan antibody capture step to capture any HCV antigen present in the testsample.

In some other embodiments, use of the pretreatment does not require sucha separation step. The entire mixture of test sample and pretreatmentreagent are contacted with an antibody specific for the targeted antigen(in this case HCV core antigen, or more particularly, HCV core antigenlipid binding domain). The pretreatment reagent employed for such anassay typically is diluted in the pretreated test sample mixture, eitherbefore or during capture by the first antibody that is used to capturethe HCV antigen. Despite such dilution, a certain amount of thepretreatment reagent may still be present in the test sample mixtureduring capture. The capture reagents may be an antibody of the presentinvention, alternatively, it may be another anti-HCV core antigenantibody or indeed it may be an antibody directed against a non-coreprotein antigen of HCV (e.g., an antibody against an envelope protein,E1, or E2 or other portion of HCV).

In one assay format, after the test sample is obtained from a subject, afirst mixture is prepared. The mixture contains the test sample beingassessed for the presence of a given antigen (e.g., in the present case,for the presence of HCV core antigen) and a first specific bindingpartner (typically an antibody that recognizes an HCV epitope), whereinthe first specific binding partner and any HCV antigen contained in thetest sample form a first antibody-antigen complex. The order in whichthe test sample and the first specific binding partner are added to formthe mixture is not critical. The first specific binding partner may beimmobilized on a solid phase, but in alternative embodiments, the firstspecific binding partner may be in a solution phase. The solid phaseused in the immunoassay (for the first specific binding partner and,optionally, the second specific binding partner) can be any solid phaseknown in the art, such as, but not limited to, a magnetic particle, abead, a test tube, a microtiter plate, a cuvette, a membrane, ascaffolding molecule, a film, a filter paper, a disc and a chip.

The methods described are amenable for adaption to systems that utilizemicroparticle technology including in automated and semi-automatedsystems wherein the solid phase comprises a microparticle. Such systemsinclude those described in pending U.S. patent application Ser. Nos.425,651 and 425,643, which correspond to published EPO applications Nos.EP 0 425 633 and EP 0 424 634, respectively, which are incorporatedherein by reference.

After the mixture containing the first specific binding partner-analytecomplex is formed, any unbound analyte is removed from the complex usingany technique known in the art. For example, the unbound analyte can beremoved by washing. Desirably, however, the first specific bindingpartner is present in excess of any analyte present in the test samplein order to optimize maximal binding of the analyte present in the testsample by the first specific binding partner.

After removal of unbound analyte, a second specific binding partner isadded to the mixture to form a first specific bindingpartner-analyte-second specific binding partner complex. The secondspecific binding partner is preferably an anti-analyte antibody thatbinds to an epitope on the analyte that differs from the epitope onanalyte bound by the first specific binding partner. Simply by way ofexample, assuming that the assay is for detection of HCV core antigen, afirst “capture” antibody is used that is specific for the DNA bindingdomain of HCV core antigen (alternatively, the first antibody is an antiHCV core antibody that is specific for the HCV core antigen lipidbinding domain, such as the antibodies described herein), once thisfirst capture antibody captures HCV core protein from the sample, asecond anti-core antigen antibody that binds the lipid binding domain ofHCV core antigen (where the first antibody bound the DNA binding domain,or alternatively, where the first antibody is first antibody is specificfor the HCV core antigen lipid binding domain, the second antibody couldbe specific for the DNA binding domain of HCV core antigen). Preferably,in such embodiments, the second specific binding partner is labeled withor contains a detectable label as described above in order to facilitatedetection of the (capture antibody-antigen-second antibody) complex.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P), an enzymatic label (such as horseradishperoxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, andthe like), a chemiluminescent label (such as acridinium esters,thioesters, or sulfonamides; luminol, isoluminol, phenanthridiniumesters, and the like), a fluorescent label (such as fluorescein (e.g.,5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)),rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zincsulfide-capped cadmium selenide), a thermometric label, or animmuno-polymerase chain reaction label. An introduction to labels,labeling procedures and detection of labels is found in Polak and VanNoorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag,N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes andResearch Chemicals (1996), which is a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. A fluorescent labelcan be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904,5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated byreference in their entireties). An acridinium compound can be used as adetectable label in a homogeneous chemiluminescent assay (see, e.g.,Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyket al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al.,Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org.Lett. 5: 3779-3782 (2003)).

A preferred acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem.63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914(1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In LuminescenceBiotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press:Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782(2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each ofwhich is incorporated herein by reference in its entirety for itsteachings regarding same).

Another preferred acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007, andpublished on Oct. 9, 2008, as U.S. Pat. App. Pub. No. 2008/0248493.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith a chemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte complexes form.Alternatively, if a second specific binding partner is used and thesecond specific binding partner is detectably labeled with achemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte-second specific bindingpartner complexes form. Any unbound specific binding partner, whetherlabeled or unlabeled, can be removed from the mixture using anytechnique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture (e.g., the source of the hydrogen peroxide beingone or more buffers or other solutions that are known to containhydrogen peroxide) before, simultaneously with, or after the addition ofan above-described acridinium compound. Hydrogen peroxide can begenerated in situ in a number of ways such as would be apparent to oneskilled in the art.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of analyte in the samplecan be quantified. Specifically, the amount of analyte in the sample isproportional to the intensity of the signal generated. The amount ofanalyte present can be quantified by comparing the amount of lightgenerated to a standard curve for analyte or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions of known concentrations of analyte by massspectroscopy, gravimetric methods, and other techniques known in theart. While the above is described with emphasis on use of an acridiniumcompound as the chemiluminescent agent, one of ordinary skill in the artcan readily adapt this description for use of other chemiluminescentagents.

Analyte immunoassays generally can be conducted using any format knownin the art, such as, but not limited to, a sandwich format.Specifically, in one immunoassay format, at least two antibodies areemployed to capture and quantify analyte, such as human analyte, or afragment thereof in a sample. More specifically, preferably, the atleast two antibodies bind to different epitopes on an analyte (or afragment thereof) forming an immune complex, which is referred to as a“sandwich.” Generally, in the immunoassays one or more antibodies can beused to capture the analyte (or a fragment thereof) in the test sample(these antibodies are frequently referred to as a “capture” antibody or“capture” antibodies) and one or more antibodies can be used to bind adetectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection antibody,” the“detection antibodies,” the “conjugate,” or the “conjugates”). Thus, inthe context of a sandwich immunoassay format, an anti-HCV core antibodyof the present invention can be used as a capture antibody, a detectionantibody, or both. For example, one anti-HCV core antibody having adomain that can bind a first epitope (e.g., the lipid binding domain ofHCV core antigen) on an analyte can be used as a capture antibody and/oranother anti-HCV core antibody having a domain that can bind a secondepitope (e.g., the DNA binding domain of HCV core antigen) can be usedas a detection antibody, or vice versa. Alternatively, one antibodyhaving a first domain that can bind an epitope on a HCV core antigen anda second antibody that binds an epitope on a different HCV antigen canbe used as a capture antibody and/or a detection antibody to detect, andoptionally quantify, two or more analytes.

Generally speaking, a sample being tested for (for example, suspected ofcontaining) analyte can be contacted with at least one capture antibody(or antibodies) and at least one detection antibody (which can be asecond detection antibody or a third detection antibody or even asuccessively numbered antibody, e.g., as where the capture and/ordetection antibody comprise multiple antibodies) either simultaneouslyor sequentially and in any order. For example, the test sample can befirst contacted with at least one capture antibody and then(sequentially) with at least one detection antibody. Alternatively, thetest sample can be first contacted with at least one detection antibodyand then (sequentially) with at least one capture antibody. In yetanother alternative, the test sample can be contacted simultaneouslywith a capture antibody and a detection antibody.

In the sandwich assay format, a sample suspected of containing analyte(or a fragment thereof) is first brought into contact with at least onefirst capture antibody under conditions that allow the formation of afirst antibody/analyte complex. If more than one capture antibody isused, a first capture antibody/analyte complex comprising two or morecapture antibodies is formed. In a sandwich assay, the antibodies, i.e.,preferably, the at least one capture antibody, are used in molar excessamounts of the maximum amount of analyte (or a fragment thereof)expected in the test sample. For example, from about 5 ug to about 1 mgof antibody per mL of buffer (e.g., microparticle coating buffer) can beused.

Competitive inhibition immunoassays, which are often used to measuresmall analytes because binding by only one antibody is required,comprise sequential and classic formats. In a sequential competitiveinhibition immunoassay a capture antibody to an analyte of interest iscoated onto a well of a microtiter plate or other solid support. Whenthe sample containing the analyte of interest is added to the well, theanalyte of interest binds to the capture antibody. After washing, aknown amount of labeled (e.g., biotin or horseradish peroxidase (HRP))analyte is added to the well. A substrate for an enzymatic label isnecessary to generate a signal. An example of a suitable substrate forHRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signalgenerated by the labeled analyte is measured and is inverselyproportional to the amount of analyte in the sample. In a classiccompetitive inhibition immunoassay an antibody to an analyte of interestis coated onto a solid support (e.g., a well of a microtiter plate).However, unlike the sequential competitive inhibition immunoassay, thesample and the labeled analyte are added to the well at the same time.Any analyte in the sample competes with labeled analyte for binding tothe capture antibody. After washing, the signal generated by the labeledanalyte is measured and is inversely proportional to the amount ofanalyte in the sample.

Optionally, prior to contacting the test sample with the at least onecapture antibody (for example, the first capture antibody), the at leastone capture antibody can be bound to a solid support, which facilitatesthe separation of the first antibody/analyte (or a fragment thereof)complex from the test sample. The substrate to which the captureantibody is bound can be any suitable solid support or solid phase thatfacilitates separation of the capture antibody-analyte complex from thesample.

Examples of solid phases or supports are well known to those of skill inthe art and include a well of a plate, such as a microtiter plate, atest tube, a porous gel (e.g., silica gel, agarose, dextran, orgelatin), a polymeric film (e.g., polyacrylamide), beads (e.g.,polystyrene beads or magnetic beads), a strip of a filter/membrane(e.g., nitrocellulose or nylon), microparticles (e.g., latex particles,magnetizable microparticles (e.g., microparticles having ferric oxide orchromium oxide cores and homo- or hetero-polymeric coats and radii ofabout 1-10 microns). The substrate can comprise a suitable porousmaterial with a suitable surface affinity to bind antigens andsufficient porosity to allow access by detection antibodies. Amicroporous material is generally preferred, although a gelatinousmaterial in a hydrated state can be used. Such porous substrates arepreferably in the form of sheets having a thickness of about 0.01 toabout 0.5 mm, preferably about 0.1 mm. While the pore size may varyquite a bit, preferably the pore size is from about 0.025 to about 15microns, more preferably from about 0.15 to about 15 microns. Thesurface of such substrates can be activated by chemical processes thatcause covalent linkage of an antibody to the substrate. Irreversiblebinding, generally by adsorption through hydrophobic forces, of theantigen or the antibody to the substrate results; alternatively, achemical coupling agent or other means can be used to bind covalentlythe antibody to the substrate, provided that such binding does notinterfere with the ability of the antibody to bind to analyte.Alternatively, the antibody can be bound with microparticles, which havebeen previously coated with streptavidin (e.g., DYNAL® Magnetic Beads,Invitrogen, Carlsbad, Calif.) or biotin (e.g., using Power-Bind™-SA-MPstreptavidin-coated microparticles (Seradyn, Indianapolis, Ind.)) oranti-species-specific monoclonal antibodies. If necessary, the substratecan be derivatized to allow reactivity with various functional groups onthe antibody. Such derivatization requires the use of certain couplingagents, examples of which include, but are not limited to, maleicanhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or more capture reagents, such asantibodies (or fragments thereof), each of which is specific foranalyte(s) can be attached to solid phases in different physical oraddressable locations (e.g., such as in a biochip configuration (see,e.g., U.S. Pat. No. 6,225,047; Int'l Patent App. Pub. No. WO 99/51773;U.S. Pat. No. 6,329,209; International Patent App. Pub. No. WO 00/56934,and U.S. Pat. No. 5,242,828). If the capture reagent is attached to amass spectrometry probe as the solid support, the amount of analytebound to the probe can be detected by laser desorption ionization massspectrometry. Alternatively, a single column can be packed withdifferent beads, which are derivatized with the one or more capturereagents, thereby capturing the analyte in a single place (see,antibody-derivatized, bead-based technologies, e.g., the xMAP technologyof Luminex (Austin, Tex.)).

After the test sample being assayed for analyte (or a fragment thereof)is brought into contact with the at least one capture antibody (forexample, the first capture antibody), the mixture is incubated in orderto allow for the formation of a first antibody (or multipleantibody)-analyte (or a fragment thereof) complex. The incubation can becarried out at a pH of from about 4.5 to about 10.0, at a temperature offrom about 2° C. to about 45° C., and for a period from at least aboutone (1) minute to about eighteen (18) hours, preferably from about 1 toabout 24 minutes, most preferably for about 4 to about 18 minutes. Theimmunoassay described herein can be conducted in one step (meaning thetest sample, at least one capture antibody and at least one detectionantibody are all added sequentially or simultaneously to a reactionvessel) or in more than one step, such as two steps, three steps, etc.

After formation of the (first or multiple) capture antibody/analytecomplex, the complex is then contacted with at least one detectionantibody under conditions which allow for the formation of a (first ormultiple) capture antibody/analyte/second detection antibody complex).While captioned for clarity as the “second” antibody (e.g., seconddetection antibody), in fact, where multiple antibodies are used forcapture and/or detection, the at least one detection antibody can be thesecond, third, fourth, etc. antibodies used in the immunoassay. If thecapture antibody/analyte complex is contacted with more than onedetection antibody, then a (first or multiple) capture antibody/analyte(or a fragment thereof)/(multiple) detection antibody complex is formed.As with the capture antibody (e.g., the first capture antibody), whenthe at least one (e.g., second and any subsequent) detection antibody isbrought into contact with the capture antibody/analyte (or a fragmentthereof) complex, a period of incubation under conditions similar tothose described above is required for the formation of the (first ormultiple) capture antibody/analyte/(second or multiple) detectionantibody complex. Preferably, at least one detection antibody contains adetectable label. The detectable label can be bound to the at least onedetection antibody (e.g., the second detection antibody) prior to,simultaneously with, or after the formation of the (first or multiple)capture antibody/analyte/(second or multiple) detection antibodycomplex. Any detectable label known in the art can be used (seediscussion above).

The detectable label can be bound to the antibodies either directly orthrough a coupling agent. An example of a coupling agent that can beused is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,hydrochloride), which is commercially available from Sigma-Aldrich, St.Louis, Mo. Other coupling agents that can be used are known in the art.Methods for binding a detectable label to an antibody are known in theart. Additionally, many detectable labels can be purchased orsynthesized that already contain end groups that facilitate the couplingof the detectable label to the antibody, such as CPSP-Acridinium Ester(i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) or SP SP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The (first or multiple) capture antibody/analyte/(second or multiple)detection antibody complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the at least one capture antibody (e.g., thefirst capture antibody) is bound to a solid support, such as a well or abead, separation can be accomplished by removing the fluid (of the testsample) from contact with the solid support. Alternatively, if the atleast first capture antibody is bound to a solid support, it can besimultaneously contacted with the analyte-containing sample and the atleast one second detection antibody to form a first (multiple)antibody/analyte/second (multiple) antibody complex, followed by removalof the fluid (test sample) from contact with the solid support. If theat least one first capture antibody is not bound to a solid support,then the (first or multiple) capture antibody/analyte/(second ormultiple) detection antibody complex does not have to be removed fromthe test sample for quantification of the amount of the label.

After formation of the labeled capture antibody/analyte/detectionantibody complex (e.g., the first capture antibody/analyte/seconddetection antibody complex), the amount of label in the complex isquantified using techniques known in the art. For example, if anenzymatic label is used, the labeled complex is reacted with a substratefor the label that gives a quantifiable reaction such as the developmentof color. If the label is a radioactive label, the label is quantifiedusing appropriate means, such as a scintillation counter. If the labelis a fluorescent label, the label is quantified by stimulating the labelwith a light of one color (which is known as the “excitationwavelength”) and detecting another color (which is known as the“emission wavelength”) that is emitted by the label in response to thestimulation. If the label is a chemiluminescent label, the label isquantified by detecting the light emitted either visually or by usingluminometers, x-ray film, high speed photographic film, a CCD camera,etc. Once the amount of the label in the complex has been quantified,the concentration of analyte or a fragment thereof in the test sample isdetermined by appropriate means, such as by use of a standard curve thathas been generated using serial dilutions of analyte or a fragmentthereof of known concentration. Other than using serial dilutions ofanalyte or a fragment thereof, the standard curve can be generatedgravimetrically, by mass spectroscopy and by other techniques known inthe art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should be about 6.0+/−0.2, themicroparticle coating buffer should be maintained at about roomtemperature (i.e., at from about 17 to about 27° C.), the microparticlecoating buffer pH should be about 6.5+/−0.2, and the microparticlediluent pH should be about 7.8+/−0.2. Solids preferably are less thanabout 0.2%, such as less than about 0.15%, less than about 0.14%, lessthan about 0.13%, less than about 0.12%, or less than about 0.11%, suchas about 0.10%.

FPIAs are based on competitive binding immunoassay principles. Afluorescently labeled compound, when excited by a linearly polarizedlight, will emit fluorescence having a degree of polarization inverselyproportional to its rate of rotation. When a fluorescently labeledtracer-antibody complex is excited by a linearly polarized light, theemitted light remains highly polarized because the fluorophore isconstrained from rotating between the time light is absorbed and thetime light is emitted. When a “free” tracer compound (i.e., a compoundthat is not bound to an antibody) is excited by linearly polarizedlight, its rotation is much faster than the correspondingtracer-antibody conjugate produced in a competitive binding immunoassay.FPIAs are advantageous over RIAs inasmuch as there are no radioactivesubstances requiring special handling and disposal. In addition, FPIAsare homogeneous assays that can be easily and rapidly performed.

In view of the above, a method of determining the presence, amount, orconcentration of HCV core (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for an HCV coreantigen (or a fragment thereof) by an assay (i) employing (i′) at leastone of an antibody, a fragment of an antibody that can bind to ananalyte, a variant of an antibody that can bind to an analyte, afragment of a variant of an antibody that can bind to an analyte, or aDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof)that can bind to an HCV core antigen, and (ii′) at least one detectablelabel and (ii) comprising comparing a signal generated by the detectablelabel as a direct or indirect indication of the presence, amount orconcentration of the HCV core antigen (or a fragment thereof) in thetest sample to a signal generated as a direct or indirect indication ofthe presence, amount or concentration of HCV core antigen (or a fragmentthereof) in a control or calibrator. The calibrator is optionally partof a series of calibrators, in which each of the calibrators differsfrom the other calibrators by the concentration of analyte.

The method can comprise (i) contacting the test sample with at least onefirst specific binding partner for HCV core (or a fragment thereof)selected from the group consisting of an antibody of the presentinvention, a fragment of such an antibody that can bind to an HCV coreantigen, a variant of an antibody that can bind to an HCV core antigen,a fragment of a variant of an antibody that can bind to an HCV coreantigen, or a DVD-Ig (or a fragment, a variant, or a fragment of avariant thereof) that can bind to an HCV core antigen so as to form afirst specific binding partner/HCV core antigen (or fragment thereof)complex, (ii) contacting the first specific binding partner/HCV coreantigen (or fragment thereof) complex with at least one second specificbinding partner for the HCV core antigen (or fragment thereof) selectedfrom the group consisting of a detectably labeled anti-HCV coreantibody, a detectably labeled fragment of an anti-HCV core antibodythat can bind to HCV core antigen, a detectably labeled variant of ananti-HCV core antibody that can bind to HCV core antigen, a detectablylabeled fragment of a variant of an anti-HCV core antibody that can bindto HCV core antigen, and a detectably labeled DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof) so as to form a firstspecific binding partner/HCV core antigen (or fragment thereof)/secondspecific binding partner complex, and (iii) determining the presence,amount or concentration of HCV core antigen in the test sample bydetecting or measuring the signal generated by the detectable label inthe first specific binding partner/HCV core antigen (or fragmentthereof)/second specific binding partner complex formed in (ii).

Alternatively, the method can comprise contacting the test sample withat least one first specific binding partner for HCV core (or a fragmentthereof) selected from the group consisting of an antibody, a fragmentof an antibody that can bind to an HCV core, a variant of an antibodythat can bind to an HCV core, a fragment of a variant of an antibodythat can bind to an HCV core, and a DVD-Ig (or a fragment, a variant, ora fragment of a variant thereof) and simultaneously or sequentially, ineither order, contacting the test sample with at least one secondspecific binding partner, which can compete with HCV core (or a fragmentthereof) for binding to the at least one first specific binding partnerand which is selected from the group consisting of a detectably labeledHCV core, a detectably labeled fragment of HCV core that can bind to thefirst specific binding partner, a detectably labeled variant of HCV corethat can bind to the first specific binding partner, and a detectablylabeled fragment of a variant of HCV core that can bind to the firstspecific binding partner. Any HCV core (or a fragment thereof) presentin the test sample and the at least one second specific binding partnercompete with each other to form a first specific binding partner/HCVcore (or fragment thereof) complex and a first specific bindingpartner/second specific binding partner complex, respectively. Themethod further comprises determining the presence, amount orconcentration of HCV core in the test sample by detecting or measuringthe signal generated by the detectable label in the first specificbinding partner/second specific binding partner complex formed in (ii),wherein the signal generated by the detectable label in the firstspecific binding partner/second specific binding partner complex isinversely proportional to the amount or concentration of HCV core in thetest sample.

The above methods can further comprise diagnosing, prognosticating, orassessing the efficacy of a therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the efficacy of a therapeutic/prophylactic treatmentof the patient from whom the test sample was obtained, the methodoptionally further comprises modifying the therapeutic/prophylactictreatment of the patient as needed to improve efficacy. The method canbe adapted for use in an automated system or a semi-automated system.

With regard to the methods of assay (and kit therefor), it may bepossible to employ commercially available anti-HCV core antibodies ormethods for production of anti-HCV core as described in the literature.Commercial supplies of various antibodies include, but are not limitedto, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech,Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for HCVcore or a fragment thereof, e.g., for detecting disease or risk ofdisease. Generally, in making such a comparison, the predetermined levelis obtained by running a particular assay a sufficient number of timesand under appropriate conditions such that a linkage or association ofHCV core presence, amount or concentration with a particular stage orendpoint of a disease, disorder or condition or with particular clinicalindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects). The HCV coremeasured can include fragments thereof, degradation products thereof,and/or enzymatic cleavage products thereof.

In particular, with respect to a predetermined level as employed formonitoring HCV disease progression and/or treatment, the amount orconcentration of analyte or a fragment thereof may be “unchanged,”“favorable” (or “favorably altered”), or “unfavorable” (or “unfavorablyaltered”). “Elevated” or “increased” refers to an amount or aconcentration in a test sample that is higher than a typical or normallevel or range (e.g., predetermined level), or is higher than anotherreference level or range (e.g., earlier or baseline sample). The term“lowered” or “reduced” refers to an amount or a concentration in a testsample that is lower than a typical or normal level or range (e.g.,predetermined level), or is lower than another reference level or range(e.g., earlier or baseline sample). The term “altered” refers to anamount or a concentration in a sample that is altered (increased ordecreased) over a typical or normal level or range (e.g., predeterminedlevel), or over another reference level or range (e.g., earlier orbaseline sample).

The typical or normal level or range for HCV core antigen is defined inaccordance with standard practice. Because the levels of HCV core insome instances will be very low, a so-called altered level or alterationcan be considered to have occurred when there is any net change ascompared to the typical or normal level or range, or reference level orrange, that cannot be explained by experimental error or samplevariation. Thus, the level measured in a particular sample will becompared with the level or range of levels determined in similar samplesfrom a so-called normal subject. In this context, a “normal subject” isan individual with no detectable disease, for example, and a “normal”(sometimes termed “control”) patient or population is/are one(s) thatexhibit(s) no detectable disease, respectively, for example.Furthermore, given that HCV core is not routinely found at a high levelin the majority of the human population, a “normal subject” can beconsidered an individual with no substantial detectable increased orelevated amount or concentration of HCV core, and a “normal” (sometimestermed “control”) patient or population is/are one(s) that exhibit(s) nosubstantial detectable increased or elevated amount or concentration ofHCV core. An “apparently normal subject” is one in which HCV core hasnot yet been or currently is being assessed. The level of an HCV core issaid to be “elevated” when the HCV core is normally undetectable (e.g.,the normal level is zero, or within a range of from about 25 to about 75percentiles of normal populations), but is detected in a test sample, aswell as when the HCV core is present in the test sample at a higher thannormal level. Thus, inter alia, the disclosure provides a method ofscreening for a subject having, or at risk of having, a particulardisease, disorder, or condition. The method of assay can also involvethe assay of other markers and the like.

Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing a HCV disease,disorder or condition. Specifically, such a method can comprise thesteps of:

(a) determining the concentration or amount in a test sample from asubject of HCV core (or a fragment thereof) (e.g., using the methodsdescribed herein, or methods known in the art); and

(b) comparing the concentration or amount of HCV core (or a fragmentthereof) determined in step (a) with a predetermined level, wherein, ifthe concentration or amount of HCV core determined in step (a) isfavorable with respect to a predetermined level, then the subject isdetermined not to have or be at risk for a given disease, disorder orcondition. However, if the concentration or amount of HCV coredetermined in step (a) is unfavorable with respect to the predeterminedlevel, then the subject is determined to have or be at risk for a givendisease, disorder or condition.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of:

(a) determining the concentration or amount in a test sample from asubject of HCV core;

(b) determining the concentration or amount in a later test sample fromthe subject of HCV core; and

(c) comparing the concentration or amount of HCV core as determined instep (b) with the concentration or amount of HCV core determined in step(a), wherein if the concentration or amount determined in step (b) isunchanged or is unfavorable when compared to the concentration or amountof HCV core determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened. By comparison, ifthe concentration or amount of HCV core as determined in step (b) isfavorable when compared to the concentration or amount of HCV core asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved.

Optionally, the method further comprises comparing the concentration oramount of HCV core as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of HCVcore as determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

In still other embodiments, any of the assays described herein formonitoring presence or levels of HCV core antigen can advantageously becombined with other assays that also determine HCV infection. Forexample, any of the HCV core determining methods of the invention mayfurther comprise determining the level of another HCV antigen or HCVantibody directed to an antigen other than core protein, including butnot limited to determining the presence of HCV Core, E1, E2, NS2, NS3,NS4a, NS4b and NS5.

Still further, the methods can be used to monitor treatment in a subjectreceiving treatment with one or more pharmaceutical compositions.Specifically, such methods involve providing a first test sample from asubject before the subject has been administered one or morepharmaceutical compositions. Next, the concentration or amount in afirst test sample from a subject of HCV core is determined (e.g., usingthe methods described herein or as known in the art). After theconcentration or amount of HCV core is determined, optionally theconcentration or amount of HCV core is then compared with apredetermined level. If the concentration or amount of HCV core asdetermined in the first test sample is lower than the predeterminedlevel, then the subject is not treated with one or more pharmaceuticalcompositions. However, if the concentration or amount of HCV core asdetermined in the first test sample is higher than the predeterminedlevel, then the subject is treated with one or more pharmaceuticalcompositions for a period of time. The period of time that the subjectis treated with the one or more pharmaceutical compositions can bedetermined by one skilled in the art (for example, the period of timecan be from about seven (7) days to about two years, preferably fromabout fourteen (14) days to about one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of HCV core is determined in thesecond or subsequent test sample is determined (e.g., using the methodsdescribed herein or as known in the art). The concentration or amount ofHCV core as determined in each of the second and subsequent test samplesis then compared with the concentration or amount of HCV core asdetermined in the first test sample (e.g., the test sample that wasoriginally optionally compared to the predetermined level). If theconcentration or amount of HCV core as determined in step (c) isfavorable when compared to the concentration or amount of HCV core asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved, and the subject shouldcontinue to be administered the one or pharmaceutical compositions ofstep (b). However, if the concentration or amount determined in step (c)is unchanged or is unfavorable when compared to the concentration oramount of HCV core as determined in step (a), then the disease in thesubject is determined to have continued, progressed or worsened, and thesubject should be treated with a higher concentration of the one or morepharmaceutical compositions administered to the subject in step (b) orthe subject should be treated with one or more pharmaceuticalcompositions that are different from the one or more pharmaceuticalcompositions administered to the subject in step (b). Specifically, thesubject can be treated with one or more pharmaceutical compositions thatare different from the one or more pharmaceutical compositions that thesubject had previously received to decrease or lower said subject's HCVcore level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained.

When used to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3days, about 4 days, about 5 days, about 6 days or about 7 days), and theinitial assay likewise is generally done within a shorter timeframe,e.g., about minutes, hours or days of the onset of the disease orcondition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the above assays can be performed using a first test sampleobtained from a subject where the first test sample is obtained from onesource, such as urine, serum or plasma. Optionally, the above assays canthen be repeated using a second test sample obtained from the subjectwhere the second test sample is obtained from another source. Forexample, if the first test sample was obtained from urine, the secondtest sample can be obtained from serum or plasma. The results obtainedfrom the assays using the first test sample and the second test samplecan be compared. The comparison can be used to assess the status of adisease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from a given disease,disorder or condition will benefit from treatment. In particular, thedisclosure relates to HCV core companion diagnostic methods andproducts. Thus, the method of “monitoring the treatment of disease in asubject” as described herein further optimally also can encompassselecting or identifying candidates for therapy.

Thus, in particular embodiments, the disclosure also provides a methodof determining whether a subject having, or at risk for, a givendisease, disorder or condition is a candidate for therapy. Generally,the subject is one who has experienced some symptom of a given disease,disorder or condition or who has actually been diagnosed as having, orbeing at risk for, a given disease, disorder or condition, and/or whodemonstrates an unfavorable concentration or amount of HCV core or afragment thereof, as described herein.

The method optionally comprises an assay as described herein, where HCVcore is assessed before and following treatment of a subject with one ormore pharmaceutical compositions (e.g., particularly with apharmaceutical related to a mechanism of action involving HCV core),with immunosuppressive therapy, or by immunoabsorption therapy, or whereHCV core is assessed following such treatment and the concentration orthe amount of HCV core is compared against a predetermined level. Anunfavorable concentration of amount of HCV core observed followingtreatment confirms that the subject will not benefit from receivingfurther or continued treatment, whereas a favorable concentration oramount of HCV core observed following treatment confirms that thesubject will benefit from receiving further or continued treatment. Thisconfirmation assists with management of clinical studies, and provisionof improved patient care.

The method of assay also can be used to identify a compound thatameliorates a given disease, disorder or condition. For example, a cellthat expresses HCV core can be contacted with a candidate compound. Thelevel of expression of HCV core in the cell contacted with the compoundcan be compared to that in a control cell using the method of assaydescribed herein.

In yet another detection method, each of the binding proteins asdescribed herein can be employed in the detection of HCV antigens infixed tissue sections, as well as fixed cells by immunohistochemicalanalysis.

In addition, these binding proteins can be bound to matrices similar toCNBr-activated Sepharose and used for the affinity purification ofspecific HCV proteins from cell cultures, or biological tissues such asblood and liver.

The monoclonal antibodies as described herein can also be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications. In addition, as discussed herein throughout the antibodiesalso could be used in the production of DVD-Ig molecules.

The monoclonal antibodies or fragments thereof can be providedindividually to detect HCV core antigens. It is contemplated thatcombinations of the monoclonal antibodies (and fragments thereof)provided herein also may be used together as components in a mixture or“cocktail” of at least one anti-HCV core antibody as described hereinwith antibodies to other HCV regions, each having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesas described herein, which are directed to HCV core protein, and othermonoclonal antibodies to other antigenic determinants of the HCV genome.Examples of other monoclonal antibodies useful for these contemplatedcocktails include those to HCV C-100, HCV 33C, HCV CORE, HCV NS5 and/orHCV putative ENV, which are disclosed in, for example, U.S. Ser. No.07/610,175 entitled MONOCLONAL ANTIBODIES TO HEPATITIS C VIRUS ANDMETHOD FOR USING SAME, U.S. Ser. No. 07/610,175 entitled MONOCLONALANTIBODIES TO HCV 33C PROTEINS AND METHODS FOR USING SAME, U.S. Ser. No.07/648,475 entitled MONOCLONAL ANTIBODIES TO PUTATIVE HCV ENVELOPEREGION AND METHODS FOR USING SAME, U.S. Ser. No. 07/648,473 entitledMONOCLONAL ANTIBODIES TO HCV CORE PROTEINS AND METHODS FOR USING SAMEand in co-filed patent application entitled MONOCLONAL ANTIBODIES TO HCVNS5 PROTEIN AND METHODS FOR USING SAME, U.S. Ser. No. 07/748,563, all ofwhich enjoy common ownership and are incorporated herein by reference.This cocktail of monoclonal antibodies as described herein would be usedin the assay formats detailed herein in place of the monoclonal antibodyto HCV core, and thus would be able to detect the HCV core and other HCVantigens.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to HCV core or other HCV proteinsused in the assay, such as HCV C-100 protein, HCV 33C protein, HCV ENV,HCV E2/NS1 or HCV NS5 protein. The polyclonal antibody used preferablyis of mammalian origin; human, goat, rabbit or sheep anti-HCV polyclonalantibody can be used. Most preferably, the polyclonal antibody is rabbitpolyclonal anti-HCV antibody. The polyclonal antibodies used in theassays can be used either alone or as a cocktail of polyclonalantibodies. Since the cocktails used in the assay formats are comprisedof either monoclonal antibodies or polyclonal antibodies havingdifferent HCV specificity, they would be useful for diagnosis,evaluation and prognosis of HCV infection, as well as for studying HCVprotein differentiation and specificity.

As noted elsewhere herein throughout, the test samples which can betested by the methods as described herein described herein include humanand animal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, biological fluids such as cell culture supernatants, fixedtissue specimens and fixed ceil specimens.

The indicator reagent comprises a signal-generating compound (label)that is capable of generating a measurable signal detectable by externalmeans conjugated (attached) to a specific binding member for HCV core.“Specific binding member” as used herein means a member of a specificbinding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. In addition to being an antibody member of a specificbinding pair for HCV core, the indicator reagent also can be a member ofany specific binding pair, including either hapten-anti-hapten systemssuch as biotin or anti-biotin, avidin or biotin, a carbohydrate or alectin, a complementary nucleotide sequence, an effector or a receptormolecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or anenzyme, and the like. An immunoreactive specific binding member can bean antibody, an antigen, or an antibody/antigen complex that is capableof binding either to HCV core as in a sandwich assay, to the capturereagent as in a competitive assay, or to the ancillary specific bindingmember as in an indirect assay.

The various signal generating compounds (labels) contemplated includechromogens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asacridinium, phenanthridinium and dioxetane compounds, radioactiveelements, and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase, and the like.The selection of a particular label is not critical, but it will becapable of producing a signal either by itself or in conjunction withone or more additional substances.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies as described herein areeasily adaptable. In scanning probe microscopy, in particular in atomicforce microscopy, the capture phase, for example, at least one of themonoclonal antibodies as described herein, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunnelling microscopy eliminates the need for labelsthat normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. Such a system is described in pending U.S.patent application Ser. No. 662,147, which enjoys common ownership andis incorporated herein by reference.

The use of SPM to monitor specific binding reactions can occur in manyways. In one embodiment, one member of a specific binding partner (theHCV core specific substance, which is the monoclonal antibody asdescribed herein) is attached to a surface suitable for scanning. Theattachment of the HCV core specific substance may be by adsorption to atest piece, which comprises a solid phase of a plastic or metal surface,following methods known to those of ordinary skill in the art. Or,covalent attachment of a specific binding partner (HCV core specificsubstance) to a test piece which test piece comprises a solid phase ofderivatized plastic, metal, silicon, or glass may be utilized. Covalentattachment methods are known to those skilled in the art and include avariety of means to irreversibly link specific binding partners to thetest piece. If the test piece is silicon or glass, the surface must beactivated prior to attaching the specific binding partner. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)trimethoxy silane(Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactivegroups such as amino-, vinyl, and thiol, respectively. Such activatedsurfaces can be used to link the binding partner directly (in the casesof amino or thiol) or the activated surface can be further reacted withlinkers such as glutaraldehyde, bis(succinimidyl) suberate, SPPD 9succinimidyl 3-[2-pyridyldithio]propionate), SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate), STAB(succinimidyl [4-iodoacetyl]aminobenzoate), and SMPB (succinimidyl4-[1-maleimidophenyl]butyrate) to separate the binding partner from thesurface. The vinyl group can be oxidized to provide a means for covalentattachment. It also can be used as an anchor for the polymerization ofvarious polymers such as poly acrylic acid, which can provide multipleattachment points for specific binding partners. The amino surface canbe reacted with oxidized dextrans of various molecular weights toprovide hydrophilic linkers of different size and capacity. Examples ofoxidizable dextrans include Dextran T-40 (molecular weight 40,000daltons), Dextran T-110 (molecular weight 110,000 daltons), DextranT-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight2,000,000 daltons) (all of which are available from Pharmacia,Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons (availablefrom Sigma Chemical Co., St. Louis, Mo.). Also, polyelectrolyteinteractions may be used to immobilize a specific binding partner on asurface of a test piece by using techniques and chemistries described bypending U.S. patent application Ser. Nos. 150,278, filed Jan. 29, 1988,and Ser. No. 375,029, filed Jul. 7, 1989, each of which enjoys commonownership and each of which is incorporated herein by reference. Thepreferred method of attachment is by covalent means. Followingattachment of a specific binding member, the surface may be furthertreated with materials such as serum, proteins, or other blocking agentsto minimize non-specific binding. The surface also may be scanned eitherat the site of manufacture or point of use to verify its suitability forassay purposes. The scanning process is not anticipated to alter thespecific binding properties of the test piece.

While the present disclosure expresses a preference for the use of solidphases, it is contemplated that the monoclonal antibodies as describedherein can be utilized in non-solid phase assay systems. These assaysystems are known to those skilled in the art, and are considered to bewithin the scope of the disclosure.

It is contemplated that the reagent employed for the assay can beprovided in the form of a kit with one or more containers such as vialsor bottles, with each container containing a separate reagent such as amonoclonal antibody, or a cocktail of monoclonal antibodies, detectionreagents and washing reagents employed in the assay.

The antibodies can also be used as a means of enhancing the immuneresponse. The antibodies can be administered in amount similar to thoseused for other therapeutic administrations of antibody. For example,normal immune globulin is administered at 0.02 0.1 ml/lb body weightduring the early incubation period of other viral diseases such asrabies, measles, and hepatitis B to interfere with viral entry intocells. Thus, antibodies reactive with the HCV core proteins can bepassively administered alone or in conjunction with another anti-viralagent to a host infected with an HCV to enhance the immune responseand/or the effectiveness of an antiviral drug.

When used as a means of inducing anti-HCV virus antibodies in an animal,the manner of injecting the antibody is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously orthe like in an effective concentration in a physiologically suitablediluent with or without adjuvant. One or more booster injections may bedesirable.

Kits

Also contemplated herein are kits for assaying a test sample for thepresence, amount or concentration of HCV core protein (or a fragmentthereof) in a test sample. Such a kit comprises at least one componentfor assaying the test sample for HCV core protein (or a fragmentthereof) and instructions for assaying the test sample for the HCV core(or a fragment thereof). The at least one component for assaying thetest sample for the HCV core (or a fragment thereof) can include acomposition comprising an anti-HCV core protein monoclonal antibody oran anti-HCV core protein DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof), which is optionally immobilized or capable ofbeing immobilized on a solid phase.

The kit can comprise at least one component for assaying the test samplefor HCV core protein by immunoassay, e.g., chemiluminescentmicroparticle immunoassay, and instructions for assaying the test samplefor an HCV core by immunoassay, e.g., chemiluminescent microparticleimmunoassay. For example, the kit can comprise at least one specificbinding partner for an HCV core, such as an anti-HCV core,monoclonal/polyclonal antibody (or a fragment thereof that can bind tothe HCV core, a variant thereof that can bind to the HCV core, or afragment of a variant that can bind to the HCV core) or an anti-HCV coreDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),either of which can be detectably labeled. Alternatively oradditionally, the kit can comprise detectably labeled HCV core (or afragment thereof that can bind to an anti-HCV core,monoclonal/polyclonal antibody or an anti-HCV core DVD-Ig (or afragment, a variant, or a fragment of a variant thereof)), which cancompete with any HCV core in a test sample for binding to an anti-HCVcore, monoclonal/polyclonal antibody (or a fragment thereof that canbind to the HCV core, a variant thereof that can bind to the HCV core,or a fragment of a variant that can bind to the HCV core) or an anti-HCVcore DVD-Ig (or a fragment, a variant, or a fragment of a variantthereof), either of which can be immobilized on a solid support. The kitcan comprise a calibrator or control, e.g., isolated or purified HCVcore. The kit can comprise at least one container (e.g., tube,microtiter plates or strips, which can be already coated with a firstspecific binding partner, for example) for conducting the assay, and/ora buffer, such as an assay buffer or a wash buffer, either one of whichcan be provided as a concentrated solution, a substrate solution for thedetectable label (e.g., an enzymatic label), or a stop solution.Preferably, the kit comprises all components, i.e., reagents, standards,buffers, diluents, etc., which are necessary to perform the assay. Theinstructions can be in paper form or computer-readable form, such as adisk, CD, DVD, or the like.

Any antibodies, such as an anti-HCV core antibody or an anti-HCV coreDVD-Ig, or tracer can incorporate a detectable label as describedherein, such as a fluorophore, a radioactive moiety, an enzyme, abiotin/avidin label, a chromophore, a chemiluminescent label, or thelike, or the kit can include reagents for carrying out detectablelabeling. The antibodies, calibrators and/or controls can be provided inseparate containers or pre-dispensed into an appropriate assay format,for example, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, enzyme substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instruments for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, a solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip.

The kit (or components thereof), as well as the method of determiningthe presence, amount or concentration of an HCV core in a test sample byan assay, such as an immunoassay as described herein, can be adapted foruse in a variety of automated and semi-automated systems (includingthose wherein the solid phase comprises a microparticle), as described,e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commerciallymarketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) asARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., an anti-HCV core,monoclonal/polyclonal antibody (or a fragment thereof, a variantthereof, or a fragment of a variant thereof) or an anti-HCV core DVD-Ig(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) is attached; either way, sandwich formation and HCV corereactivity can be impacted), and the length and timing of the capture,detection and/or any optional wash steps. Whereas a non-automatedformat, such as an ELISA, may require a relatively longer incubationtime with sample and capture reagent (e.g., about 2 hours), an automatedor semi-automated format (e.g., ARCHITECT®, Abbott Laboratories) mayhave a relatively shorter incubation time (e.g., approximately 18minutes for ARCHITECT®). Similarly, whereas a non-automated format, suchas an ELISA, may incubate a detection antibody, such as the conjugatereagent, for a relatively longer incubation time (e.g., about 2 hours),an automated or semi-automated format (e.g., ARCHITECT®) may have arelatively shorter incubation time (e.g., approximately 4 minutes forthe ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays Immunosensors andtheir methods of manufacture and operation in single-use test devicesare described, for example in, U.S. Pat. No. 5,063,081, U.S. Patent App.Pub. No. 2003/0170881, U.S. Patent App. Pub. No. 2004/0018577, U.S.Patent App. Pub. No. 2005/0054078, and U.S. Patent App. Pub. No.2006/0160164, which are incorporated in their entireties by referencefor their teachings regarding same.

In particular, with regard to the adaptation of an HCV core assay to theI-STAT® system, the following configuration is preferred. Amicrofabricated silicon chip is manufactured with a pair of goldamperometric working electrodes and a silver-silver chloride referenceelectrode. On one of the working electrodes, polystyrene beads (0.2 mmdiameter) with immobilized anti-HCV core, monoclonal/polyclonal antibody(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) or anti-HCV core DVD-Ig (or a fragment thereof, a variantthereof, or a fragment of a variant thereof), are adhered to a polymercoating of patterned polyvinyl alcohol over the electrode. This chip isassembled into an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising a specific binding partner foran HCV core, such as an anti-HCV core, monoclonal/polyclonal antibody(or a fragment thereof, a variant thereof, or a fragment of a variantthereof that can bind the HCV core) or an anti-HCV core DVD-Ig (or afragment thereof, a variant thereof, or a fragment of a variant thereofthat can bind the HCV core), either of which can be detectably labeled.Within the fluid pouch of the cartridge is an aqueous reagent thatincludes p-aminophenol phosphate.

In operation, a sample suspected of containing an HCV core is added tothe holding chamber of the test cartridge, and the cartridge is insertedinto the I-STAT® reader. After the specific binding partner for an HCVcore has dissolved into the sample, a pump element within the cartridgeforces the sample into a conduit containing the chip. Here it isoscillated to promote formation of the sandwich. In the penultimate stepof the assay, fluid is forced out of the pouch and into the conduit towash the sample off the chip and into a waste chamber. In the final stepof the assay, the alkaline phosphatase label reacts with p-aminophenolphosphate to cleave the phosphate group and permit the liberatedp-aminophenol to be electrochemically oxidized at the working electrode.Based on the measured current, the reader is able to calculate theamount of HCV core in the sample by means of an embedded algorithm andfactory-determined calibration curve.

It further goes without saying that the methods and kits as describedherein necessarily encompass other reagents and methods for carrying outthe immunoassay. For instance, encompassed are various buffers such asare known in the art and/or which can be readily prepared or optimizedto be employed, e.g., for washing, as a conjugate diluent, microparticlediluent, and/or as a calibrator diluent. An exemplary conjugate diluentis ARCHITECT® conjugate diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.), which comprises a buffer containingMES, other salt, a protein blocker, and an antimicrobial agent.Additionally, as described in U.S. Patent Application No. 61/142,048filed Dec. 31, 2008, improved signal generation may be obtained, e.g.,in an I-Stat cartridge format, using a nucleic acid sequence linked tothe signal antibody as a signal amplifier.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1

Animal Immunizations.

Female CAF1/J and RBF/DnJ mice (both from The Jackson Laboratory, BarHarbor, Me.) were immunized on weeks 0, 4 and 10 with 50 μg of an HCVcore peptide corresponding to amino acids (all numbering per HCV-1)134-171 covalently linked to BSA (ALRZ-8 immunogen).

HCV core peptide-BSA was prepared by AnaSpec, Inc. (Fremont, Calif.).The immunogen peptide was emulsified in Complete or Incomplete AdjuliteFreund's Adjuvant (Pacific Immunology, Ramona, Calif.). CompleteFreund's adjuvant was used for the primary immunization and IncompleteFreund's adjuvant for the second and third immunizations. Each inoculumwas prepared by first diluting the HCV peptide-BSA to the appropriateconcentration in sterile saline (0.9% sodium chloride), adding an equalvolume of adjuvant and then mixing by passing back and forth between twosyringes via a 3-way stopcock until a thick, stable emulsion was formed.Sera samples were taken 10-14 days following the 3rd immunization. Onthe 4^(th) and 3^(rd) days prior to B cell harvest, RBF/DnJ mice #306and 315 were administered 50 μg peptide-BSA diluted in sterile saline.This inoculum was delivered into the body cavity near the spleen.

ALRZ-8 immunogen (SEQ ID NO: 576)Ac-MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGC-BSA.

Example 2

Screening of Mouse Sera for Antigen Immunoreactivity.

Mouse sera samples collected 7-10 days after their final immunizationwere first tested in a 96-well micro titer enzyme immunoassay (EIA) forreactivity to each of three synthetic (Anaspec, Inc.) carboxy-terminalbiotinylated HCV core peptides. The peptides used for screening werederived from the immunogen sequence described in Example 1 and had thefollowing designations and sequences: Peptide 1 (all numbering perHCV-1), amino acids 134-151: MGYIPLVGAPLGGAARALAHG (SEQ ID NO:573);Peptide 2, amino acids 141-161: GAPLGGAARALAHGVRVLEDG (SEQ ID NO:574),Peptide 3, amino acids 151-171, LAHGVRVLEDGVNYATGNLPG (SEQ ID NO:575).Assay plates (NUNC Corporation, Naperville, Ill.) were coated with 100μL/well of sheep anti-mouse IgG Fc specific antibody (JacksonImmunoResearch, West Grove, Pa.) diluted to 2 μg/mL inphosphate-buffered saline (PBS). Plates were incubated at 37 deg C. forabout 2 hours and then at about 21 deg C. for about 2 hours. The captureantibody was then removed and 200 μL/well of blocking solution (3% w/v[weight/volume] bovine serum albumin (BSA) and 0.5% v/v [volume/volume]polysorbate-20 diluted in PBS added. The plates were incubated for about30 minutes and then washed with distilled water. Next, serial dilutions(in block solution) of the mouse sera or a positive control were addedto the assay plates (100 μL/well), incubated for between 2 and 20 hoursand then washed with dH2O. Next, 100 μL/well of normal serum solution(NSS; block solution containing 2% v/v normal mouse serum) was added foradditional blocking. This solution helps to prevent non-specific bindingin the assay well. The plates were incubated for about 30 minutes andthen washed with dH2O. Subsequently, 100 μL/well of a 224 nM solution ofeach peptide was added to the assay wells for a brief incubation, afterwhich the plates were washed with dH2O (sera samples were tested forreactivity to individual peptides, rather than a mixture of all three).Next, 100 μL/well horse radish peroxidase labeled streptavidin (JacksonImmunoResearch) diluted to 200 ng/mL in blocking solution was added,allowed to incubate for about 30 minutes and then the plates washed;o-phenylenediamine substrate (OPD) was used as the chromagen to generatesignal, and the reaction was quenched using 1 N sulfuric acid. Signalwas read at a wavelength of 492 nm.

Example 3

Screening of Mouse Sera for Relative Affinity.

Relative affinity testing was completed for each sera sample—peptidecombination for which a strong signal was seen in the previous assay. Todetermine the relative affinity of each serum sample for the individualcore peptides, samples were tested for reactivity to limitingconcentrations of each biotin labeled peptide. The assay format wasidentical to that described above, except that instead of preparingserial dilutions of mouse sera test samples, each sample was prepared ata single dilution, in blocking solution. Additionally, the individualpeptides were tested at varying concentrations, beginning with a 500 nMsolution in blocking solution followed by ten log 2 dilutions, also inblocking solution. Binding curves were generate and used to determinerelative affinity for each sera sample. Based on these results, RBF/DnJmice #306 and 315 were chosen for B cell fusion.

Example 4

Mouse Splenocyte Fusion.

On the day of fusion, the mice were euthanized and their splenocyteswere harvested and placed into Iscoves Modified Dulbeccos Medium (IMDM)supplemented with Pen Strep (Invitrogen Corporation). A cell fusion wasperformed as described by Kohler and Milstein (Nature 1975; 256:495-7).Each mouse spleen was placed into a petri dish containing IMDM. Thesplenocytes were perfused out of each spleen using a syringe containingIMDM and a cell scraper. All splenocytes from mouse #306 and 315 wereisolated and pooled in a 50 ml centrifuge tube, then counted using ahemocytometer with trypan blue dye exclusion to determine viability.Approximately 8.0×10⁸ total cells at 89% viability were recovered fromthese spleens. Approximately 7.6×10⁶ cells/ml were estimated to beB-cells based on their physical appearance under the microscope.Approximately 5 mL of this cell suspension was used for a first fusionexperiment (fusion 208A), and the remaining cells were processed usingmagnetic activated cell sorting (MACS) and a Pan B-cell isolation kit(Miltenyi Biotech) to enrich the cell population for B-cells and depleteother cell types. Approximately 6.7×10⁸ total cells were incubated withthe Pan B-cell biotin labeled antibody cocktail followed by theanti-biotin micobeads per manufacturer's instructions. The cellsuspension/microbead mixture was washed by centrifugation and passedover a Miltenyi Biotech LS column contained within a magnetic field.B-cells flow freely through the column and other cell types are retainedin the column. The columns were washed 3 times with PBS containing 2%FBS to wash out all B-cells. The B-cell suspension was centrifuged andthe pellet was resuspended in IMDM, and then counted using ahemocytometer. Approximately 1.4×10⁸ B-cells were recovered from theenrichment procedure. Approximately 7.0×10′ B-cells from that suspensionwere used for a second fusion experiment (fusion 208B) and the remainingB-cells were cryopreserved for later usage.

The unenriched splenocytes from the spleens (˜3.8×10⁷B-cells for fusion208A) and the enriched B-cell pool (˜7.0×10⁷B-cells for fusion 208B)were washed by centrifugation in separate tubes and the cell pelletswere re-suspended in IMDM. These splenocytes were mixed with an equalnumber of NS/0 myeloma cells and centrifuged into a pellet. The fusionwas accomplished by exposing the splenocytes and NS/0 cells to 50%Polyethylene glycol (PEG) (American Type Culture Collection—MolecularWeight 1300-1600) in HSFM. One mL of the PEG solution was added to eachcell pellet over 30 seconds, followed by one additional minute ofincubation. The PEG and cell pellet was diluted by slowly adding 30 mLof IMDM over 30 seconds. The fused cells were then removed fromsuspension by centrifugation and decanting the supernatant. The cellpellet from each fusion (208A and 208B) was re-suspended into ˜250 mL ofIMDM supplemented with ˜10% FBS (Hyclone Laboratories), HAT(Hypoxanthine, Aminopterin, Thymidine) (Sigma Laboratories), HTSupplement (Invitrogen Corporation), BM Condimed H1 (Roche AppliedScience), Cholesterol and L-Glutamine (Invitrogen Corporation) in orderto select for hybridomas. The fused cells were seeded into T162 cultureflasks containing the HAT medium and cultured in bulk for approximately48 hours at 37° C. with 5% CO2. Following 48 hours of HAT selection, thebulk culture was centrifuged and the pellet was re-suspended intosemi-solid tissue culture medium. The semi-solid tissue culture mediumconsisted of a 50% mixture of 2×IMDM (Invitrogen) with Clone Matrix(Molecular Devices) supplemented with 10% FBS, HT Supplement,Penn/Strep, L-Glutamine, and anti-mouse IgG-FITC Clone Detect (MolecularDevices). The semi-solid culture plates were allowed to incubate for7-10 days before colony selection on the ClonepixFL (Molecular Devices).A colony grown in the semi-solid medium was considered a clone becausethe single cell initiating it had not been allowed to move and mix withother cells during growth, but all cell lines of interest will besubcloned at a later date to ensure clonality. An immunoprecipitationreaction occurs between the antibody being produced by the colony andthe goat anti-mouse IgG Fc-FITC that fluoresces. The brighter thefluorescence signal observed, the more antibody being produced. Colonieswere analyzed for fluorescence on the ClonepixFL and the ones with thebrightest fluorescent signal were selected for automated transfer to 96well tissue culture plates containing IMDM supplemented with 10% FBS, HTsupplement, cholesterol, L-Glutamine, and Pen Strep. The 96 well tissueculture plates were allowed to grow for 3 to 7 days at 37° C. prior tosupernatant screening for antibody production.

Example 5

Hybridoma Screening and Selection Using Peptides.

Cell supernatant samples were analyzed for anti-HCV antibodies by EIA.Sheep anti-mouse IgG Fc (Jackson Immunoresearch) was coated on 96 wellmicro-titer EIA plates at 1 μg/mL. After the capture reagent had beencoated on the solid phase, remaining solution was removed and the plateswere blocked using 3% BSA in PBS. The wells were washed with distilledwater and cell supernatants were added to the blocked plates and allowedto incubate at room temperature for at least one hour. The anti-mouseIgG Fc captures the anti-HCV mouse antibody from the supernatant.Following the incubation, the plates were washed using distilled water.A 3% normal mouse serum in BSA block solution was added to all wells andincubated at room temperature for 30 minutes to block any unbound sheepanti-mouse IgG Fc capture sites coated on the plate. The wells werewashed with distilled water and a mixture of the biotinylated HCVpeptides described in Example 2 (i.e. corresponding to amino acids134-154, 141-161, and 151-171 of HCV-1), each at 100 ng/mL, was addedand incubated for 30 minutes at room temperature. Following thisincubation, the biotinylated antigens were washed from the plates usingdistilled water and streptavidin-HRPO (Jackson Immunoresearch) dilutedto approximately 200 ng/mL was added to the plates and allowed toincubate for 30 minutes. The plates were washed with distilled water ando-phenylenediamine substrate was used as the chromagen to generatesignal. Plates were read at 492 nm and the results were analyzed. Wellswere considered positive if they had an EIA signal at least 3 timesgreater than background. Positive wells were expanded to 24 well platesin IMDM supplemented with 10% FBS, HT supplement, cholesterol, andL-Glutamine.

Following 5-14 days of growth, the 24 well cultures were evaluated byEIA in the same manner as previously described, except the supernatantsamples were titrated against each of the biotinylated HCV core peptidesindividually and BSA to identify clones that might bind nonspecificallyto the peptides or the blocking protein. The 24 well cultures generatingsignal at least 5 times greater than the average BSA background value of0.08 OD units with at least one of the screening peptides wereconsidered positive and selected for further evaluation. Values arelisted in Table 1.

TABLE 1 Reactivity to Reactivity to Reactivity to Clone # HCV bt-134-154HCV bt-141-161 HCV bt-151-171 BSA (Pep) HCV 208A-1006 0.15 3.56 0.070.08 HCV 208A-1007 0.07 3.53 0.08 0.07 HCV 208A-1015 0.07 0.42 0.11 0.08HCV 208A-1022 0.08 1.63 0.08 0.09 HCV 208A-1032 0.12 3.73 0.09 0.08 HCV208A-1064 0.10 3.50 0.12 0.07 HCV 208A-1081 0.06 3.52 0.06 0.05 HCV208A-110 0.07 2.88 0.11 0.06 HCV 208A-122 0.06 2.65 0.09 0.06 HCV208A-126 0.20 2.08 0.10 0.10 HCV 208A-133 0.10 3.68 0.13 0.06 HCV208A-134 2.06 0.08 0.06 0.07 HCV 208A-147 0.67 0.07 0.06 0.20 HCV208A-152 0.08 3.55 0.09 0.07 HCV 208A-158 0.10 2.70 0.09 0.07 HCV208A-159 0.15 3.77 0.19 0.08 HCV 208A-160 0.11 3.28 0.09 0.09 HCV208A-194 0.07 0.20 0.06 0.07 HCV 208A-207 1.24 0.17 0.08 0.07 HCV208A-208 0.09 3.22 0.29 0.10 HCV 208A-210 0.06 1.15 0.05 0.06 HCV208A-222 0.06 2.70 0.08 0.07 HCV 208A-227 0.08 0.24 0.43 0.07 HCV208A-230 0.08 0.42 0.07 0.07 HCV 208A-264 0.05 0.07 0.37 0.09 HCV208A-286 0.11 2.62 0.11 0.06 HCV 208A-293 0.08 2.42 0.11 0.07 HCV208A-312 0.22 3.70 0.06 0.06 HCV 208A-334 0.05 3.07 0.05 0.05 HCV208A-352 1.08 0.07 0.06 0.07 HCV 208A-367 0.08 1.79 0.10 0.08 HCV208A-381 0.07 3.69 0.12 0.08 HCV 208A-382 0.10 0.07 0.43 0.08 HCV208A-393 0.06 1.04 0.06 0.05 HCV 208A-422 0.05 0.84 0.10 0.07 HCV208A-427 0.20 3.82 0.11 0.11 HCV 208A-442 0.08 3.04 0.06 0.06 HCV208A-460 0.09 2.69 0.10 0.07 HCV 208A-470 0.08 0.10 0.08 0.08 HCV208A-489 0.06 3.11 0.07 0.06 HCV 208A-493 0.09 3.60 0.07 0.07 HCV208A-557 0.05 2.41 0.06 0.05 HCV 208A-558 0.12 0.95 0.14 0.10 HCV208A-562 0.21 3.71 0.12 0.09 HCV 208A-575 0.07 1.55 0.13 0.12 HCV208A-576 0.08 3.05 0.09 0.06 HCV 208A-584 0.89 0.10 0.06 0.08 HCV208A-603 0.07 1.35 0.09 0.07 HCV 208A-604 0.05 2.27 0.06 0.05 HCV208A-605 0.22 3.71 0.16 0.11 HCV 208A-638 0.07 2.96 0.09 0.06 HCV208A-641 0.10 0.93 0.11 0.09 HCV 208A-658 0.06 1.10 0.08 0.20 HCV208A-692 3.67 0.38 0.07 0.06 HCV 208A-695 0.08 3.82 0.11 0.07 HCV208A-719 0.07 3.60 0.09 0.06 HCV 208A-736 3.24 0.12 0.06 0.07 HCV208A-741 1.20 3.37 0.06 0.07 HCV 208A-744 0.12 3.88 0.10 0.08 HCV208A-759 0.16 3.95 0.14 0.08 HCV 208A-768 0.11 3.28 0.09 0.09 HCV208A-774 0.14 3.53 0.12 0.07 HCV 208A-793 0.07 2.96 0.10 0.07 HCV208A-807 0.79 0.07 0.06 0.06 HCV 208A-826 3.28 0.11 0.11 0.10 HCV208A-828 0.07 3.35 0.10 0.07 HCV 208A-830 0.07 2.88 0.08 0.06 HCV208A-850 0.07 3.49 0.10 0.06 HCV 208A-863 0.07 4.00 0.08 0.06 HCV208A-874 0.05 0.73 0.06 0.05 HCV 208A-877 0.06 1.21 0.05 0.06 HCV208A-879 0.09 0.17 0.79 0.19 HCV 208A-920 0.17 4.00 0.08 0.08 HCV208A-926 0.06 3.32 0.07 0.09 HCV 208A-938 0.09 3.49 0.09 0.10 HCV208A-939 3.33 0.17 0.05 0.05 HCV 208A-967 0.52 0.73 0.18 0.07 HCV208A-982 0.08 3.40 0.06 0.07 HCV 208A-983 3.03 0.40 0.05 0.05 HCV208B-1024 0.08 0.75 0.15 0.10 HCV 208B-1029 0.07 0.51 0.10 0.06 HCV208B-1043 0.16 0.12 3.88 0.07 HCV 208B-1070 0.07 3.50 0.05 0.05 HCV208B-1072 0.09 2.28 0.09 0.12 HCV 208B-109 0.08 3.46 0.11 0.07 HCV208B-1094 0.12 3.28 0.13 0.10 HCV 208B-1096 0.34 0.13 3.56 0.08 HCV208B-131 0.13 0.18 3.89 0.10 HCV 208B-141 1.66 0.09 0.06 0.06 HCV208B-174 0.08 1.26 0.07 0.08 HCV 208B-178 0.10 0.07 1.69 0.07 HCV208B-181 0.12 2.44 0.07 0.06 HCV 208B-183 0.31 0.14 0.09 0.11 HCV208B-189 0.55 0.15 3.95 0.09 HCV 208B-207 0.19 0.09 0.50 0.07 HCV208B-214 0.16 0.07 0.60 0.06 HCV 208B-230 0.41 0.12 0.08 0.08 HCV208B-251 0.11 2.35 0.41 0.10 HCV 208B-281 1.48 0.16 0.12 0.07 HCV208B-309 0.11 2.34 0.09 0.09 HCV 208B-319 0.17 3.43 0.13 0.13 HCV208B-327 0.07 0.05 0.49 0.05 HCV 208B-348 0.07 0.20 0.07 0.07 HCV208B-353 0.07 2.47 0.07 0.07 HCV 208B-395 0.54 0.13 3.69 0.11 HCV208B-408 0.81 0.20 0.16 0.06 HCV 208B-409 0.09 3.21 0.14 0.07 HCV208B-446 0.06 2.50 0.08 0.06 HCV 208B-457 0.75 0.66 0.24 0.17 HCV208B-471 1.62 0.11 0.08 0.10 HCV 208B-488 0.08 0.07 0.30 0.08 HCV208B-515 0.51 0.13 0.06 0.06 HCV 208B-517 0.10 0.41 0.70 0.11 HCV208B-547 0.12 0.08 2.90 0.08 HCV 208B-556 0.13 3.07 0.18 0.08 HCV208B-560 0.11 3.90 0.14 0.07 HCV 208B-589 0.27 0.10 2.85 0.12 HCV208B-591 0.08 3.10 0.07 0.07 HCV 208B-602 0.20 1.87 0.20 0.07 HCV208B-608 0.19 0.37 0.13 0.07 HCV 208B-612 3.05 3.04 0.15 0.08 HCV208B-616 0.10 0.71 0.11 0.09 HCV 208B-617 0.45 0.51 0.13 0.10 HCV208B-646 0.15 3.94 0.18 0.10 HCV 208B-652 0.21 0.55 0.10 0.09 HCV208B-672 0.08 2.79 0.10 0.08 HCV 208B-739 1.13 0.11 0.07 0.08 HCV208B-742 0.08 0.44 0.07 0.09 HCV 208B-750 0.10 3.41 0.12 0.09 HCV208B-762 0.07 0.65 0.05 0.05 HCV 208B-765 0.10 0.64 0.16 0.08 HCV208B-778 0.11 0.06 0.23 0.06 HCV 208B-780 0.07 1.02 0.17 0.10 HCV208B-788 0.09 0.49 0.10 0.16 HCV 208B-793 0.09 0.72 0.09 0.13 HCV208B-796 0.07 3.03 0.07 0.09 HCV 208B-822 0.08 1.35 0.07 0.05 HCV208B-826 0.53 1.35 0.08 0.07 HCV 208B-853 0.09 0.74 0.12 0.13 HCV208B-860 0.12 0.73 0.12 0.07 HCV 208B-862 0.21 3.28 0.10 0.09 HCV208B-894 0.07 0.09 0.48 0.07 HCV 208B-909 0.09 3.11 0.11 0.07 HCV208B-911 0.09 0.09 0.71 0.10 HCV 208B-922 0.93 0.11 0.11 0.09 HCV208B-952 0.07 0.64 0.07 0.09 HCV 208B-954 0.06 3.43 0.06 0.06 HCV208B-956 0.10 0.85 0.05 0.05 HCV 208B-960 0.09 3.38 0.11 0.08 HCV208B-982 0.20 1.10 0.13 0.09

Example 6

Cloning and Expression of Recombinant HCV Corel-169.

The nucleotide sequence encoding amino acids 1-169 of HCV-1 was codonoptimized for E. coli expression and cloned into a modified pET32avector wherein the sequence encoding a thioredoxin fusion protein waseliminated and replaced with Methionine (M). In addition, acarboxy-terminal hexahistidine tag (SEQ ID NO:580) was includedimmediately after codon 169 of HCV core to facilitate purification viaimmobilized metal affinity chromatography (IMAC). E. coli BL21(DE3)cells were transformed with purified plasmid DNA and a clone harboringthe plasmid pET-HCVCore1-169 identified. The protein expressed therefromwas designated as HCV Corel-169.

Protein expression was achieved by culturing thepET-HCVCore1-169-transformed E. coli BL21(DE3) cells in terrific broth(TB) medium. Cells were grown in a fermentor to an OD600 nm of 10 andthen induced with 1 mM IPTG and grown at 37° C. for approximately threehours until an OD600 nm of approximately 20 was obtained. Cells wereharvested by centrifugation and lysed by sonication in 25 mM Tris-HClbuffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl₂, lysozymeand benzonase. The lysate was clarified by centrifugation and theinsoluble fraction was dissolved in 25 mM Tris-HCl buffer (pH 7.4)containing 150 mM NaCl, 6 M urea, 1.0% n-dodecyl-B-D-maltoside, 1 mMDTT, and 5 mM MgCl₂. The dissolved lysate was again clarified bycentrifugation and the soluble fraction was loaded onto a HisTrap FastFlow column (GE Healthcare). The column was then washed with 25 columnvolumes of 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mMDTT, 5 mM MgCl₂, 6 M urea, 0.1% n-dodecyl-B-D-maltoside, and 10 mMimidazole. Elution was done using the same buffer and a linear gradientof imidazole (0-500 mM). Eluted fractions containing the desired proteinof interest (determined by SDS-PAGE) were pooled and dialyzed against 25mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mMMgCl₂, with and without 6 M urea and 0.1% n-dodecyl-B-D-maltoside.

HCV Core1-169 Nucleotide Sequence (SEQ ID NO: 571)Atgtctaccaacccgaaaccgcagaaaaaaaacaaacgtaacaccaaccgtcgtccgcaggacgttaaattcccgggtggtggtcagatcgttggtggtgtttacctgctgccgcgtcgtggtccgcgtctgggtgttcgtgctacgcgtaaaacctctgaacgttctcagccgcgtgggcgtcgtcagccgatcccgaaagctcgtcgtccggaaggtcgtacctgggctcagccgggttacccgtggccgctgtacggtaacgaaggttgcggttgggctggttggctgctgtctccgcgtggatctcgtccgtcttggggtccgaccgacccgcgtcgtcgttctcgtaaccttggtaaagttatcgataccctgacctgcggtttcgctgacctgatgggttacataccgctggttggagctccgctgggtggtgctgctcgtgctctggcgcatggcgtgcgtgttctggaagatggcgtcaactatgccaccgg taatctgHCV Core1-169 Amino Acid Sequence (SEQ ID NO: 572)Mstnpkpqkknkrntnrrpqdvkfpgggqivggvyllprrgprlgvratrktsersqprgrrqpipkarrpegrtwaqpgypwplygnegcgwagwllsprgsrpswgptdprrrsrnlgkvidtltcgfadlmgyiplvgaplggaara lahgvrvledgvnyatgnl.

Example 7

Hybridoma Screening Using Core Antigen.

The 24 well cultures were then evaluated by EIA for their ability bindHCV core1-169 (as described in Example 6) coated directly ontomicrotiter plates (solid phase assay). HCV core1-169 was coated onto 96well micro-titer EIA plates at 1 μg/mL. After the capture reagent hadbeen coated on the solid phase, the solution was removed and the plateswere blocked using 3% BSA in PBS. The wells were washed with distilledwater and 5 fold serial dilutions of the cell culture supernatants wereadded and allowed to incubate at room temperature for at least one hour.The plates were washed with distilled water and a HRP labeled goatanti-mouse IgG FC antibody diluted at approximately 200 ng/ml in BSAblock solution was added to the plates and allowed to incubate for 30minutes at room temperature. The plates were washed with distilled waterand o-phenylenediamine substrate was used as the chromagen to generatesignal. Plates were read at 492 nm and the results were analyzed.

Antibodies with BSA background reactivity greater than or equal to thecore 1-169 reactivity value were considered negative and not used incalculating the average BSA background value. For the remainder ofantibodies to be considered positive for core 1-169, they had to have anEIA signal of, at least, 0.50 OD units, or at least 5 times greater thanthe average BSA background signal of 0.10 OD units. Values are listed inTable 2.

TABLE 2 Solid Phase HCV Core BSA Clone # OD (A492) OD (A492) HCV208A-1006 0.75 0.09 HCV 208A-1007 0.39 0.06 HCV 208A-1015 0.16 0.08 HCV208A-1022 0.09 0.07 HCV 208A-1032 1.27 0.08 HCV 208A-1064 0.94 0.08 HCV208A-1081 1.25 0.06 HCV 208A-110 1.60 0.08 HCV 208A-122 1.36 0.06 HCV208A-126 0.69 0.24 HCV 208A-133 1.73 0.07 HCV 208A-134 0.10 0.07 HCV208A-147 0.08 0.10 HCV 208A-152 1.31 0.07 HCV 208A-158 1.16 0.06 HCV208A-159 0.57 0.35 HCV 208A-160 1.65 0.08 HCV 208A-194 0.08 0.07 HCV208A-207 0.11 0.06 HCV 208A-208 0.77 0.28 HCV 208A-210 0.87 0.06 HCV208A-222 1.06 0.07 HCV 208A-227 0.06 0.08 HCV 208A-230 0.08 0.06 HCV208A-264 0.09 0.07 HCV 208A-286 1.20 0.08 HCV 208A-293 1.41 0.07 HCV208A-312 1.39 0.07 HCV 208A-334 1.28 0.09 HCV 208A-352 0.10 0.08 HCV208A-367 0.88 0.07 HCV 208A-381 1.43 0.13 HCV 208A-382 0.08 0.07 HCV208A-393 0.24 0.07 HCV 208A-422 1.68 1.10 HCV 208A-427 0.62 0.11 HCV208A-442 1.61 0.06 HCV 208A-460 1.68 0.07 HCV 208A-470 0.08 0.07 HCV208A-489 1.37 0.07 HCV 208A-493 1.58 0.06 HCV 208A-557 1.40 0.07 HCV208A-558 0.40 0.25 HCV 208A-562 1.29 0.07 HCV 208A-575 0.12 0.06 HCV208A-576 1.13 0.06 HCV 208A-584 0.07 0.06 HCV 208A-603 0.25 0.07 HCV208A-604 1.27 0.06 HCV 208A-605 0.55 0.06 HCV 208A-638 1.07 0.06 HCV208A-641 0.20 0.07 HCV 208A-658 0.83 0.07 HCV 208A-692 0.07 0.06 HCV208A-695 0.61 0.17 HCV 208A-719 1.12 0.06 HCV 208A-736 0.07 0.07 HCV208A-741 1.20 0.06 HCV 208A-744 1.57 0.07 HCV 208A-759 0.36 0.22 HCV208A-768 1.12 0.07 HCV 208A-774 1.41 0.06 HCV 208A-793 1.24 0.06 HCV208A-807 0.14 0.10 HCV 208A-826 0.09 0.06 HCV 208A-828 1.49 0.07 HCV208A-830 1.16 0.06 HCV 208A-850 1.18 0.06 HCV 208A-863 1.55 0.07 HCV208A-874 0.45 0.06 HCV 208A-877 0.90 0.07 HCV 208A-879 0.32 0.30 HCV208A-920 1.14 0.07 HCV 208A-926 1.14 0.09 HCV 208A-938 0.83 0.13 HCV208A-939 0.07 0.06 HCV 208A-967 1.08 1.30 HCV 208A-982 1.25 0.07 HCV208A-983 0.12 0.06 HCV 208B-1024 0.60 0.40 HCV 208B-1029 0.07 0.07 HCV208B-1043 0.86 0.15 HCV 208B-1070 1.24 0.08 HCV 208B-1072 0.89 0.07 HCV208B-109 0.62 0.15 HCV 208B-1094 0.90 0.17 HCV 208B-1096 0.49 0.15 HCV208B-131 0.74 0.06 HCV 208B-141 0.07 0.08 HCV 208B-174 0.15 0.06 HCV208B-178 0.89 0.07 HCV 208B-181 0.47 0.07 HCV 208B-183 0.84 0.85 HCV208B-189 0.49 0.11 HCV 208B-207 0.06 0.08 HCV 208B-214 0.09 0.06 HCV208B-230 0.08 0.09 HCV 208B-251 0.79 0.08 HCV 208B-281 0.09 0.07 HCV208B-309 0.77 0.07 HCV 208B-319 0.50 0.11 HCV 208B-327 0.14 0.08 HCV208B-348 0.90 0.67 HCV 208B-353 0.30 0.06 HCV 208B-395 0.50 0.12 HCV208B-408 0.09 0.08 HCV 208B-409 0.52 0.13 HCV 208B-446 0.78 0.24 HCV208B-457 0.13 0.11 HCV 208B-471 0.24 0.12 HCV 208B-488 0.34 0.07 HCV208B-515 0.06 0.07 HCV 208B-517 0.79 0.07 HCV 208B-547 0.46 0.13 HCV208B-556 0.74 0.36 HCV 208B-560 1.07 0.07 HCV 208B-589 0.68 0.17 HCV208B-591 0.72 0.08 HCV 208B-602 0.14 0.06 HCV 208B-608 0.07 0.06 HCV208B-612 0.18 0.06 HCV 208B-616 0.19 0.15 HCV 208B-617 0.93 1.21 HCV208B-646 1.24 0.07 HCV 208B-652 0.10 0.07 HCV 208B-672 1.77 0.79 HCV208B-739 0.19 0.17 HCV 208B-742 0.29 0.08 HCV 208B-750 0.98 0.07 HCV208B-762 1.62 1.49 HCV 208B-765 0.15 0.12 HCV 208B-778 0.10 0.08 HCV208B-780 1.05 0.97 HCV 208B-788 0.21 0.21 HCV 208B-793 0.85 0.42 HCV208B-796 0.49 0.14 HCV 208B-822 0.22 0.06 HCV 208B-826 0.65 0.08 HCV208B-853 0.10 0.06 HCV 208B-860 0.12 0.08 HCV 208B-862 0.49 0.12 HCV208B-894 0.07 0.07 HCV 208B-909 0.66 0.23 HCV 208B-911 1.18 1.17 HCV208B-922 0.07 0.08 HCV 208B-952 0.16 0.07 HCV 208B-954 1.10 0.07 HCV208B-956 0.12 0.07 HCV 208B-960 1.16 0.09 HCV 208B-982 0.46 0.08

Example 8

Hybridoma Screening Via Core Antigen Capture Assay.

The cell lines that were identified as positive at the 24 well stage bypeptide-based EIA (Example 5) or HCV Corel-169 solid phase immunoassay(Example 7) were expanded for cryopreservation, followed by generationof high-density spent-cell supernatant. The high density exhaustedsupernatant from fusion 208A and 208B cell lines were tested for theirability to detect HCV Corel-169 captured from solution by a monoclonalantibody (14-153-229, U.S. Pat. No. 7,858,752) directed against anepitope within the nucleic acid binding domain of HCV Core (e.g. aminoacids 1-125), also known as Domain 1. An anti-domain 1 monoclonalantibody was coated on the solid phase of 96 well micro-titer EIA platesat 1 μg/mL. After the capture reagent had been coated on the solidphase, it was removed and the plates were blocked for 30 minutes at roomtemperature using a 5×PBS buffer containing 2% fish gelatin, 0.5%Tween20, and 0.1% n-Dodecyl-N,N-Dimethylamine-N-Oxide (Affymetrix). Theplates were washed with distilled water and a 50 ng/ml solution ofCorel-169 antigen diluted in the fish gelatin/detergent solution wasadded to all wells and allowed to incubate for at least 30 minutes atroom temperature. The wells were washed with distilled water and cellsupernatants were titrated down the blocked plates and allowed toincubate at room temperature for at least 30 minutes at roomtemperature. The plates were washed with distilled water and a HRPlabeled goat anti-mouse IgG FC antibody diluted to approximately 200ng/ml in BSA block solution was added to the plates and allowed toincubate for 30 minutes at room temperature. The plates were washed withdistilled water and o-phenylenediamine substrate was used as thechromagen to generate signal. Plates were read at 492 nm and the resultswere analyzed. Antibodies were considered positive for Corel-169 if theyhad EIA signal of, at least, 0.50 OD units, or at least 5 times greaterthan the average BSA background signal of 0.10 OD units. Values arelisted in Table 3.

TABLE 3 Captured HCV Core BSA Clone # OD (A492) OD (A492) HCV 208A-10060.97 0.10 HCV 208A-1007 1.25 0.13 HCV 208A-1015 0.15 0.14 HCV 208A-10220.33 0.22 HCV 208A-1032 1.53 0.08 HCV 208A-1064 1.57 0.09 HCV 208A-10811.95 0.09 HCV 208A-110 1.87 0.07 HCV 208A-122 2.10 0.07 HCV 208A-1261.20 0.07 HCV 208A-133 1.46 0.08 HCV 208A-134 0.13 0.09 HCV 208A-1470.14 0.08 HCV 208A-152 1.60 0.06 HCV 208A-158 1.84 0.08 HCV 208A-1591.29 0.08 HCV 208A-160 1.54 0.08 HCV 208A-194 0.14 0.14 HCV 208A-2070.41 0.09 HCV 208A-208 1.77 0.07 HCV 208A-210 0.87 0.08 HCV 208A-2222.07 0.08 HCV 208A-227 0.09 0.08 HCV 208A-230 0.07 0.08 HCV 208A-2640.09 0.08 HCV 208A-286 2.06 0.14 HCV 208A-293 2.31 0.07 HCV 208A-3122.07 0.08 HCV 208A-334 1.90 0.10 HCV 208A-352 0.53 0.08 HCV 208A-3671.41 0.08 HCV 208A-381 2.18 0.07 HCV 208A-382 0.11 0.08 HCV 208A-3930.07 0.09 HCV 208A-422 0.18 0.11 HCV 208A-427 1.84 0.08 HCV 208A-4420.36 0.14 HCV 208A-460 1.50 0.08 HCV 208A-470 0.09 0.09 HCV 208A-4891.46 0.07 HCV 208A-493 1.44 0.08 HCV 208A-557 2.39 0.08 HCV 208A-5580.23 0.18 HCV 208A-562 2.15 0.07 HCV 208A-575 0.30 0.10 HCV 208A-5761.44 0.07 HCV 208A-584 1.10 1.20 HCV 208A-603 1.55 0.09 HCV 208A-6041.75 0.07 HCV 208A-605 2.21 0.08 HCV 208A-638 2.24 0.08 HCV 208A-6410.11 0.10 HCV 208A-658 0.91 0.08 HCV 208A-692 0.57 0.09 HCV 208A-6951.77 0.09 HCV 208A-719 1.68 0.08 HCV 208A-736 0.32 0.10 HCV 208A-7411.90 0.08 HCV 208A-744 1.62 0.10 HCV 208A-759 0.63 0.08 HCV 208A-7682.29 0.08 HCV 208A-774 1.91 0.09 HCV 208A-793 1.19 0.08 HCV 208A-8070.09 0.08 HCV 208A-826 0.17 0.08 HCV 208A-828 1.55 0.08 HCV 208A-8302.09 0.08 HCV 208A-850 1.50 0.09 HCV 208A-863 1.88 0.08 HCV 208A-8740.18 0.10 HCV 208A-877 1.25 0.08 HCV 208A-879 0.15 0.11 HCV 208A-9201.82 0.08 HCV 208A-926 1.83 0.08 HCV 208A-938 1.66 0.08 HCV 208A-9390.31 0.09 HCV 208A-967 0.18 0.15 HCV 208A-982 1.44 0.09 HCV 208A-9830.64 0.09 HCV 208B-1024 0.31 0.12 HCV 208B-1029 0.14 0.15 HCV 208B-10431.21 0.07 HCV 208B-1070 1.25 0.08 HCV 208B-1072 1.45 0.08 HCV 208B-1091.28 0.10 HCV 208B-1094 1.51 0.08 HCV 208B-1096 1.75 0.07 HCV 208B-1311.52 0.08 HCV 208B-141 0.17 0.11 HCV 208B-174 0.75 0.09 HCV 208B-1781.09 0.08 HCV 208B-181 1.65 0.07 HCV 208B-183 0.11 0.10 HCV 208B-1891.81 0.12 HCV 208B-207 0.07 0.08 HCV 208B-214 0.08 0.08 HCV 208B-2300.13 0.09 HCV 208B-251 1.39 0.10 HCV 208B-281 0.11 0.12 HCV 208B-3091.23 0.08 HCV 208B-319 1.45 0.08 HCV 208B-327 0.11 0.09 HCV 208B-3480.21 0.23 HCV 208B-353 0.91 0.10 HCV 208B-395 1.95 0.08 HCV 208B-4080.09 0.10 HCV 208B-409 1.34 0.10 HCV 208B-446 1.08 0.08 HCV 208B-4570.09 0.07 HCV 208B-471 0.35 0.08 HCV 208B-488 0.10 0.08 HCV 208B-5150.08 0.08 HCV 208B-517 0.97 0.12 HCV 208B-547 1.62 0.07 HCV 208B-5561.35 0.18 HCV 208B-560 1.30 0.07 HCV 208B-589 1.29 0.08 HCV 208B-5911.66 0.08 HCV 208B-602 0.24 0.07 HCV 208B-608 0.09 0.08 HCV 208B-6121.20 0.08 HCV 208B-616 0.09 0.08 HCV 208B-617 0.07 0.08 HCV 208B-6461.29 0.08 HCV 208B-652 0.10 0.09 HCV 208B-672 0.99 0.08 HCV 208B-7390.88 0.09 HCV 208B-742 0.12 0.08 HCV 208B-750 1.53 0.08 HCV 208B-7620.28 0.21 HCV 208B-765 0.36 0.44 HCV 208B-778 0.08 0.07 HCV 208B-7800.10 0.08 HCV 208B-788 0.09 0.07 HCV 208B-793 0.17 0.20 HCV 208B-7961.19 0.09 HCV 208B-822 0.12 0.11 HCV 208B-826 0.72 0.41 HCV 208B-8530.08 0.09 HCV 208B-860 0.09 0.09 HCV 208B-862 1.07 0.07 HCV 208B-8940.09 0.09 HCV 208B-909 0.39 0.10 HCV 208B-911 0.14 0.17 HCV 208B-9220.08 0.08 HCV 208B-952 0.09 0.08 HCV 208B-954 1.53 0.07 HCV 208B-9560.14 0.12 HCV 208B-960 1.19 0.08 HCV 208B-982 0.99 0.08

Example 9

Determination of Anti-HCV Core Antibody Binding Kinetics.

The affinities/kinetics of the anti-HCV core peptide 134-171 monoclonalantibodies were determined using a Biacore 4000 instrument (GEHealthcare Bio-Sciences AB, Uppsala, Sweden).

First, after pre-treating a CM5 Series S biosensor chip (GE Healthcare)with duplicate injections of 100 mM HCl, 50 mM NaOH, and 0.1% SDS, arabbit anti-mouse IgG Capture Biosensor was created by amine-couplingrabbit anti-mouse IgG antibody (GE Healthcare, Piscataway, N.J.) onSpots 1, 2, 4, and 5 in all four flow cells of the biosensor chip viaEDC/NHS/ethanolamine chemistry provided in an Amine Coupling Kit (GEHealthcare). Clarified anti-HCV Core antibody exhausted hybridomasupernatants and HCV Core peptide were diluted into a filtered buffercomposed of 10×HBS-EP+ buffer (GE Healthcare; hereinafter “runningbuffer”) diluted 10-fold into distilled H₂O, supplemented with 0.1% BSAand 0.1% CM-Dextran, and 0.2 μm filtered. Each HCV Core antibodysupernatant was diluted 1:1 with running buffer and 0.2 μm filteredagain. A 53 amino acid custom peptide (ALRZ-9 peptide, Anaspec, Fremont,Calif.) was chemically synthesized to contain HCV Core residues 134-171and a carboxy-terminal tetanus toxoid (TT) immunogenic T-cell epitopepeptide (Eur. J. Immunol. (1989), 19:2237-2242) with the terminal aminoand carboxy groups acetylated and amidated, respectively. Thelyophilized HCV core 134-171-tetanus toxoid synthetic peptide immunogenwas diluted in distilled water to a stock concentration of 0.7 or 1mg/mL and further diluted into running buffer to concentrations ofeither 0.457 to 3,000 nM or 0.412 to 2,700 nM, both using a 3-folddilution series. All antigen solutions were 0.2 μm filtered prior touse.

The HCV Core 134-171-TT peptide procedure was as follows: 10 μL of HCVCore antibody was separately injected over Spots 1 and 5 in all fourflow cells at 10 μL/minute. After all the spots contained capturedantibody, the flow rate was increased to 30 μL/minute and the biosensorwas equilibrated at this new flow rate for 2 minutes; then, a 3 minuteinjection (90 μL) of HCV Core peptide followed by 4 minutes of runningbuffer. All biosensor surfaces were regenerated with one 30 μL injectionof 10 mM glycine, pH 1.7 (GE Healthcare), at a flow rate of 10μL/minute. All concentrations were tested in duplicate. The bindingkinetics (association and dissociation) were monitored via sensorgramsduring antigen injection followed by running buffer. The sensorgramswere double-referenced and fit to a 1:1 binding model using Biacore 4000Evaluation software (GE Healthcare Bio-Sciences AB) to determineassociation and dissociation rates, as well as overall K_(D). Kineticand affinity values are listed in Table 4. If values are not present,then either binding kinetics could not be determined or the antibody didnot interact with the HCV Core 134-171-TT peptide in this assay.

ALRZ-9 peptide (SEQ ID NO: 577)Ac-MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGQYIKANSKF IGITEL-NH2

TABLE 4 Binding Interactions with HCV Core 134-171-TT Peptide Clone # ka(1/Ms) kd (Vs) KD (M) HCV 208A-1006 1.8E+06 6.2E−04 3.5E−10 HCV208A-1007 1.8E+06 7.4E−04 4.1E−10 HCV 208A-1022 1.6E+05 2.5E−03 1.6E−08HCV 208A-1032 1.2E+06 5.5E−04 4.5E−10 HCV 208A-1064 1.6E+06 3.4E−042.1E−10 HCV 208A-1081 1.3E+06 5.7E−04 4.5E−10 HCV 208A-110 1.7E+065.5E−04 3.3E−10 HCV 208A-122 1.8E+06 2.1E−04 1.2E−10 HCV 208A-1331.2E+06 5.0E−04 4.1E−10 HCV 208A-134 1.8E+04 3.8E−05 2.1E−09 HCV208A-152 7.7E+05 4.0E−04 5.2E−10 HCV 208A-158 1.3E+06 4.4E−04 3.3E−10HCV 208A-159 1.4E+06 6.5E−04 4.5E−10 HCV 208A-160 1.6E+06 6.1E−043.8E−10 HCV 208A-207 3.1E+05  <1E−05  <3.2E−11   HCV 208A-208 1.6E+065.4E−04 3.4E−10 HCV 208A-222 1.1E+06 3.5E−04 3.1E−10 HCV 208A-2862.0E+06 2.5E−04 1.2E−10 HCV 208A-293 2.1E+06 2.3E−04 1.1E−10 HCV208A-312 2.1E+06 2.6E−04 1.3E−10 HCV 208A-334 1.7E+06 5.7E−04 3.4E−10HCV 208A-352 1.0E+05 5.4E−05 5.2E−10 HCV 208A-367 1.5E+06 5.9E−043.9E−10 HCV 208A-381 2.0E+06 2.7E−04 1.3E−10 HCV 208A-427 1.2E+065.6E−04 4.5E−10 HCV 208A-460 1.3E+06 5.6E−04 4.4E−10 HCV 208A-4891.3E+06 6.6E−04 4.9E−10 HCV 208A-493 1.4E+06 5.7E−04 4.1E−10 HCV208A-557 2.4E+06 2.1E−04 9.1E−11 HCV 208A-562 1.7E+06 6.6E−04 4.0E−10HCV 208A-576 1.2E+06 5.8E−04 4.9E−10 HCV 208A-603 1.5E+06 5.6E−043.9E−10 HCV 208A-604 1.3E+06 5.8E−04 4.5E−10 HCV 208A-605 8.7E+053.7E−04 4.3E−10 HCV 208A-638 2.7E+06 5.3E−04 2.0E−10 HCV 208A-6952.0E+06 7.1E−04 3.5E−10 HCV 208A-719 1.9E+06 5.7E−04 3.0E−10 HCV208A-736 2.2E+04 1.7E−05 7.6E−10 HCV 208A-741 1.7E+06 6.9E−04 4.1E−10HCV 208A-744 1.3E+06 6.0E−04 4.5E−10 HCV 208A-768 1.1E+06 3.6E−043.3E−10 HCV 208A-774 1.3E+06 5.9E−04 4.5E−10 HCV 208A-793 1.8E+066.6E−04 3.7E−10 HCV 208A-826 1.8E+04 7.4E−05 4.2E−09 HCV 208A-8281.4E+06 5.7E−04 4.2E−10 HCV 208A-830 3.4E+06 5.4E−04 1.6E−10 HCV208A-850 1.6E+06 6.0E−04 3.9E−10 HCV 208A-863 1.3E+06 6.1E−04 4.6E−10HCV 208A-877 1.1E+06 2.4E−03 2.2E−09 HCV 208A-920 1.5E+06 5.6E−043.9E−10 HCV 208A-926 1.7E+06 5.6E−04 3.3E−10 HCV 208A-938 1.6E+065.2E−04 3.2E−10 HCV 208A-939 2.5E+04 2.2E−04 9.0E−09 HCV 208A-9821.3E+06 6.2E−04 4.9E−10 HCV 208A-983 9.7E+04 4.6E−05 4.6E−10 HCV208B-1024 1.0E+06 1.4E−02 1.5E−08 HCV 208B-1043 1.1E+06 6.0E−04 5.4E−10HCV 208B-1070 1.6E+06 5.2E−04 3.2E−10 HCV 208B-1072 1.3E+06 5.5E−044.2E−10 HCV 208B-109 1.4E+06 5.1E−04 3.7E−10 HCV 208B-1094 1.5E+062.5E−04 1.6E−10 HCV 208B-1096 1.3E+06 6.0E−04 4.6E−10 HCV 208B-1311.3E+06 1.7E−03 1.3E−09 HCV 208B-141 3.1E+04 1.6E−04 5.3E−09 HCV208B-174 7.6E+05 1.8E−04 2.5E−10 HCV 208B-181 2.0E+06 3.6E−04 1.8E−10HCV 208B-189 8.8E+05 1.5E−04 1.7E−10 HCV 208B-251 1.1E+06 6.5E−045.8E−10 HCV 208B-281 2.2E+03 2.5E−04 9.6E−08 HCV 208B-309 1.2E+066.8E−04 5.7E−10 HCV 208B-319 2.2E+06 2.8E−04 1.3E−10 HCV 208B-3951.3E+06 4.7E−04 3.6E−10 HCV 208B-409 1.9E+06 2.8E−04 1.5E−10 HCV208B-446 2.1E+06 2.8E−04 1.4E−10 HCV 208B-471 2.2E+05 5.5E−05 2.5E−10HCV 208B-547 1.6E+06 4.9E−03 3.2E−09 HCV 208B-556 1.5E+06 6.0E−044.0E−10 HCV 208B-560 1.1E+06 2.6E−04 2.3E−10 HCV 208B-589 9.6E+056.1E−04 6.3E−10 HCV 208B-591 2.2E+06 2.0E−04 9.3E−11 HCV 208B-6121.8E+05 6.5E−05 3.5E−10 HCV 208B-646 1.8E+06 6.0E−04 3.4E−10 HCV208B-672 2.2E+06 3.4E−04 1.6E−10 HCV 208B-739 2.8E+05 5.5E−05 2.0E−10HCV 208B-750 2.0E+06 2.6E−04 1.3E−10 HCV 208B-796 1.5E+06 6.8E−044.6E−10 HCV 208B-862 1.4E+06 1.0E−04 7.0E−11 HCV 208B-954 1.6E+065.5E−04 3.4E−10 HCV 208B-960 1.6E+06 5.6E−04 3.5E−10 HCV 208B-9822.2E+06 8.0E−04 3.5E−10

Example 10

BIACore Antibody Binding Pair Analysis with Nucleic Acid Binding DomainmAbs

The ability of the anti-HCV core peptide 134-171 monoclonal antibodiesto form antibody binding pairs with anti-HCV Core C11-3, C11-7, C11-9,and C11-14 (U.S. Pat. No. 6,727,092; Morota, et al, J. Virol. Meth.,2009, 157:8-14) antibodies and recombinant HCV Corel-169 antigen wasdetermined using a Biacore 4000 instrument (GE Healthcare Bio-SciencesAB). First, after pre-treating a CM5 Series S biosensor chip (GEHealthcare) with duplicate injections of 100 mM HCl, 50 mM NaOH, and0.1% SDS, a rabbit anti-mouse IgG Capture Biosensor was created byamine-coupling rabbit anti-mouse IgG antibody (GE Healthcare,Piscataway, N.J.) on Spots 1, 2, 4, and 5 in all four flow cells of thebiosensor chip via EDC/NHS/ethanolamine chemistry provided in an AmineCoupling Kit (GE Healthcare).

Clarified anti-HCV Core (peptide aa 134-171) antibody exhaustedhybridoma supernatants, recombinant HCV Corel-169 antigen, 3 differentpurified mouse monoclonal IgG representing isotypes IgG1, IgG2a, andIgG2b non-reactive to HCV Core used as blocking reagents, and anti-HCVCore C11-3, C11-7, C11-9, and C11-14 mAbs were diluted into a filteredrunning buffer (hereinafter “running buffer”) composed of 10×PBS buffer(GE Healthcare) diluted 5-fold into distilled H₂O, supplemented with 3mM EDTA, 0.1% BSA, 0.1% CM-Dextran, 0.1%n-Dodecyl-N,N-Dimethylamine-N-Oxide, an extra 500 mM NaCl, and 0.2 μmfiltered. Each HCV Core antibody supernatant was diluted 1:1, therecombinant HCV Corel-169 antigen was diluted to 500 nM per thecalculated dimer molecular weight (39,453 Da), the anti-HCV Core C11-3,C11-7, C11-9, and C11-14 purified monoclonal antibodies wereindividually diluted to 20 μg/mL, and the 3 mouse IgG blocking reagentswere all diluted as a pool with each isotype having a concentration ofat least 100 μg/mL in running buffer. All dilutions were 0.2 μm filteredprior to use.

The HCV Core antibody-antigen-antibody sandwich procedure was asfollows. 20 μL of the HCV Core C11 antibodies were injected over Spots 1and 5 in all four flow cells at 10 μL/minute: C11-3 in flow cell 1,C11-7 in flow cell 2, C11-9 in flow cell 3, and c11-14 in flow cell 4.The flow rate was increased to 30 μL/minute and the remaining availableanti-mouse IgG binding sites on the biosensor were blocked with themouse IgG1, IgG2a, and IgG2b isotype pool by injecting 60 μL over Spots1 and 2 and then 60 μL over Spots 4 and 5 in all flow cells. 60 μL ofHCV Corel-169 antigen was injected over Spot 1 and then another 60 μLover Spot 5 in all flow cells. 60 μL of one HCV Core antibody dilutedsupernatant was inject over Spots 1 and 2 over all flow cells andanother diluted supernatant over Spots 4 and 5 in all flow cells. Theflow rate was decreased 10 μL/minute and all biosensor surfaces wereregenerated with one 30 μL injection of 10 mM glycine, pH 1.7 (GEHealthcare).

Using the Biacore 4000 Evaluation Epitope Mapping software module (GEHealthcare Bio-Sciences AB), the binding response for each C11 antibody,antigen, and HCV Core antibody supernatant was determined after eachinjection and used to calculate an expected response reference valueusing the dimeric antigen and antibody (150,000 Da) molecular weights.An expected percent binding value was determined using individualsamples versus the reference value. Any expected percent binding valuesthat were greater than 5.0 were considered positive for the ability toform an antibody sandwich with the recombinant HCV Corel-169 antigen.Expected percent values are listed in Table 5.

TABLE 5 Secondary Conjugate Antibody Primary Capture Antibody Anti-HCVCore 134-171 Anti-HCV Core antibody antibodies C11-3 C11-7 C11-9 C11-14HCV 208A-1006 2.4 3.1 <1.0 2.5 HCV 208A-1007 4.4 6.1 2.7 4.2 HCV208A-1015 <1.0 <1.0 <1.0 <1.0 HCV 208A-1022 <1.0 1.3 <1.0 <1.0 HCV208A-1032 19.5 26.7 15.7 21.8 HCV 208A-1064 16.3 19.6 10.5 16.3 HCV208A-1081 19.0 25.9 14.9 20.8 HCV 208A-110 15.7 21.1 9.4 14.8 HCV208A-122 17.3 19.7 9.6 14.8 HCV 208A-126 1.2 2.0 <1.0 1.3 HCV 208A-1339.8 13.9 5.8 9.3 HCV 208A-134 <1.0 <1.0 <1.0 <1.0 HCV 208A-147 <1.0 <1.0<1.0 <1.0 HCV 208A-152 10.0 12.5 6.9 9.8 HCV 208A-158 14.0 16.5 7.8 12.4HCV 208A-159 4.6 7.0 3.3 5.5 HCV 208A-160 15.5 20.9 8.7 13.8 HCV208A-194 <1.0 <1.0 <1.0 <1.0 HCV 208A-207 <1.0 <1.0 <1.0 <1.0 HCV208A-208 2.6 3.2 <1.0 2.5 HCV 208A-210 1.2 1.6 <1.0 <1.0 HCV 208A-22215.6 18.8 10.7 15.0 HCV 208A-227 <1.0 1.1 <1.0 <1.0 HCV 208A-230 <1.0<1.0 <1.0 <1.0 HCV 208A-264 <1.0 1.1 <1.0 <1.0 HCV 208A-286 18.9 22.113.0 18.5 HCV 208A-293 17.9 20.9 11.0 16.4 HCV 208A-312 16.3 19.7 11.816.4 HCV 208A-334 17.2 20.6 10.3 15.9 HCV 208A-352 <1.0 <1.0 <1.0 <1.0HCV 208A-367 9.7 14.5 6.1 9.3 HCV 208A-381 17.2 20.3 12.3 17.4 HCV208A-382 <1.0 1.5 <1.0 <1.0 HCV 208A-393 <1.0 <1.0 <1.0 <1.0 HCV208A-422 <1.0 <1.0 <1.0 <1.0 HCV 208A-427 15.5 22.1 11.7 16.5 HCV208A-442 <1.0 1.6 <1.0 <1.0 HCV 208A-460 17.5 24.3 12.9 18.3 HCV208A-470 <1.0 <1.0 <1.0 <1.0 HCV 208A-489 15.9 21.9 11.5 16.6 HCV208A-493 12.6 17.7 7.8 12.2 HCV 208A-557 17.7 21.4 13.3 17.6 HCV208A-558 <1.0 1.1 <1.0 <1.0 HCV 208A-562 13.8 17.1 9.5 14.8 HCV 208A-575<1.0 <1.0 <1.0 <1.0 HCV 208A-576 12.6 16.9 9.1 13.3 HCV 208A-584 <1.01.6 <1.0 <1.0 HCV 208A-603 6.7 9.1 4.0 6.8 HCV 208A-604 15.4 20.8 9.814.8 HCV 208A-605 15.9 19.3 11.9 15.8 HCV 208A-638 14.1 16.9 9.2 13.3HCV 208A-641 <1.0 <1.0 <1.0 <1.0 HCV 208A-658 1.8 2.5 <1.0 <1.0 HCV208A-692 <1.0 <1.0 <1.0 <1.0 HCV 208A-695 14.2 20.4 9.5 15.0 HCV208A-719 7.5 10.0 4.9 8.0 HCV 208A-736 <1.0 <1.0 <1.0 <1.0 HCV 208A-74119.4 22.7 14.2 21.2 HCV 208A-744 16.7 22.5 10.5 16.1 HCV 208A-759 <1.0<1.0 <1.0 <1.0 HCV 208A-768 12.7 17.1 8.8 12.6 HCV 208A-774 18.5 24.513.4 19.3 HCV 208A-793 3.8 6.2 2.5 3.9 HCV 208A-807 <1.0 <1.0 <1.0 <1.0HCV 208A-826 <1.0 1.3 <1.0 <1.0 HCV 208A-828 14.8 20.7 10.9 16.5 HCV208A-830 11.9 14.7 7.9 11.7 HCV 208A-850 7.3 9.7 4.4 7.5 HCV 208A-86318.0 24.9 12.1 18.0 HCV 208A-874 <1.0 <1.0 <1.0 <1.0 HCV 208A-877 9.412.1 5.9 10.7 HCV 208A-879 <1.0 <1.0 <1.0 <1.0 HCV 208A-920 19.3 22.913.2 19.1 HCV 208A-926 19.3 26.3 14.7 21.1 HCV 208A-938 14.1 21.0 10.415.5 HCV 208A-939 <1.0 <1.0 <1.0 <1.0 HCV 208A-967 <1.0 <1.0 <1.0 <1.0HCV 208A-982 19.2 26.3 15.0 20.6 HCV 208A-983 <1.0 <1.0 <1.0 <1.0 HCV208B-1024 10.9 14.3 7.1 12.2 HCV 208B-1029 <1.0 <1.0 <1.0 <1.0 HCV208B-1043 17.4 20.7 12.0 17.9 HCV 208B-1070 22.0 28.6 17.7 25.3 HCV208B-1072 25.0 30.2 19.3 27.4 HCV 208B-109 12.5 16.0 8.6 13.3 HCV208B-1094 24.4 28.4 19.8 27.8 HCV 208B-1096 15.1 16.9 11.4 16.3 HCV208B-131 11.4 12.4 6.8 11.6 HCV 208B-141 <1.0 <1.0 <1.0 <1.0 HCV208B-174 1.6 1.5 <1.0 1.6 HCV 208B-178 <1.0 1.7 <1.0 <1.0 HCV 208B-1817.4 10.0 5.4 8.1 HCV 208B-183 <1.0 <1.0 <1.0 <1.0 HCV 208B-189 2.3 2.9<1.0 2.7 HCV 208B-207 <1.0 <1.0 <1.0 <1.0 HCV 208B-214 <1.0 <1.0 <1.0<1.0 HCV 208B-230 <1.0 <1.0 <1.0 <1.0 HCV 208B-251 16.6 20.1 12.3 18.2HCV 208B-281 <1.0 <1.0 <1.0 <1.0 HCV 208B-309 13.6 17.5 10.2 15.5 HCV208B-319 6.0 8.5 4.2 6.3 HCV 208B-327 <1.0 <1.0 <1.0 <1.0 HCV 208B-348<1.0 1.5 <1.0 <1.0 HCV 208B-353 1.9 2.6 <1.0 2.4 HCV 208B-395 7.6 9.45.2 7.8 HCV 208B-408 <1.0 <1.0 <1.0 <1.0 HCV 208B-409 1.8 2.7 <1.0 2.0HCV 208B-446 10.0 12.2 7.8 10.3 HCV 208B-457 <1.0 <1.0 <1.0 <1.0 HCV208B-471 <1.0 <1.0 <1.0 <1.0 HCV 208B-488 <1.0 <1.0 <1.0 <1.0 HCV208B-515 <1.0 <1.0 <1.0 <1.0 HCV 208B-517 <1.0 <1.0 <1.0 <1.0 HCV208B-547 7.2 8.3 4.9 8.2 HCV 208B-556 11.5 15.3 8.3 12.7 HCV 208B-56010.6 15.5 8.3 12.0 HCV 208B-589 9.4 11.7 6.7 9.9 HCV 208B-591 19.1 23.015.1 21.7 HCV 208B-602 1.3 1.8 <1.0 <1.0 HCV 208B-608 <1.0 <1.0 <1.0<1.0 HCV 208B-612 3.8 4.4 <1.0 3.1 HCV 208B-616 <1.0 <1.0 <1.0 <1.0 HCV208B-617 <1.0 <1.0 <1.0 <1.0 HCV 208B-646 22.0 30.2 17.8 25.6 HCV208B-652 <1.0 1.3 <1.0 <1.0 HCV 208B-672 2.6 2.8 <1.0 2.7 HCV 208B-739<1.0 <1.0 <1.0 <1.0 HCV 208B-742 <1.0 <1.0 <1.0 <1.0 HCV 208B-750 22.827.3 17.2 24.1 HCV 208B-762 <1.0 <1.0 <1.0 <1.0 HCV 208B-765 <1.0 <1.0<1.0 <1.0 HCV 208B-778 <1.0 <1.0 <1.0 <1.0 HCV 208B-780 <1.0 <1.0 <1.0<1.0 HCV 208B-788 <1.0 <1.0 <1.0 <1.0 HCV 208B-793 <1.0 <1.0 <1.0 <1.0HCV 208B-796 20.2 27.7 17.3 25.2 HCV 208B-822 <1.0 <1.0 <1.0 <1.0 HCV208B-826 <1.0 <1.0 <1.0 <1.0 HCV 208B-853 <1.0 <1.0 <1.0 <1.0 HCV208B-860 <1.0 <1.0 <1.0 <1.0 HCV 208B-862 11.2 15.2 8.9 12.6 HCV208B-894 <1.0 <1.0 <1.0 <1.0 HCV 208B-909 1.6 2.5 <1.0 <1.0 HCV 208B-911<1.0 <1.0 <1.0 <1.0 HCV 208B-922 <1.0 <1.0 <1.0 <1.0 HCV 208B-952 <1.0<1.0 <1.0 <1.0 HCV 208B-954 23.9 32.7 18.4 26.0 HCV 208B-956 <1.0 <1.0<1.0 <1.0 HCV 208B-960 21.0 28.1 15.8 22.9 HCV 208B-982 4.5 5.4 <1.0 4.9

Example 11

Immunoglobulin Purification and Labeling.

Anti-HCV core hybridomas were expanded in Hybridoma Serum Free Medium(Invitrogen Corporation) supplemented with L-glutamine and 10% Ultra LowIgG FBS (Invitrogen Corporation) and seeded into roller bottles atapproximately 0.5×10E⁵ cells/mL. The cultures were incubated at 37° C.while rotating at approximately 1 revolution per minute for 10-14 days,or until a terminal end culture was obtained. The terminal roller bottlesupernatant was harvested and clarified with a 0.45 micron filter. Theclarified supernatant was diluted with an equal volume of 1.5 M glycine,3M NaCl buffer, pH 8.9, then loaded onto a pre-equilibrated 5 ml ProteinA column using the AKTA automated purification system(Amersham/Pharmacia/GE). The column was then washed with approximately 5column volumes of binding buffer and when a stable baseline is achieved,the mAb was eluted with 0.1 M sodium citrate buffer, pH 2.8. The IgG wasthen transferred to a desalting column and exchanges into PBS, pH7.2-7.4, and then further dialyzed in PBS pH 7.2-7.4, using 10,000molecular weight cut-off dialysis membrane (Pierce Chemical). Selectedantibodies were biotinylated by using Sulfo-NHS-LC-Biotin (Pierce) at a20-fold molar excess and incubated for 30 minutes at room temperature.Unbound biotin was removed through dialysis in PBS pH 7.2-7.4. Allbiotinylated monoclonals were tested by EIA to confirm successfullabeling.

Example 12

HCV Core Antigen Capture Assays.

Purified anti-HCV core134-171 monoclonal antibodies were evaluated fortheir ability to form binding pairs with themselves and two other domain1 monoclonal antibodies using HCV core1-169 recombinant antigen in anEIA format. Anti-HCV Domain 1 monoclonal antibodies, C11-7 and C11-9,and anti-HCV core134-171 monoclonals were coated onto microtiter platesat approximately 1000 ng/ml and allowed to incubate overnight at 2-8degrees C. After the capture reagent had been coated on the solid phase,the plates were blocked using a 5×PBS buffer containing 2% fish gelatin,0.5 Tween 20, and 0.1% n-dodecyl-N,N-dimethylamine-N-oxide. The wellswere washed with distilled water and purified core 1-169 antigen wasadded to the blocked plates in serial dilutions from 50 to 0.78 ng/mldiluted in fish gelatin block, and then allowed to incubate at roomtemperature for approximately 30 minutes. The wells were washed withdistilled water and biotin labeled anti-HCV core monoclonals were addedto the plates at concentrations ranging from 100 to 5000 ng/ml, and thenincubated for 30 minutes at room temperature. The plates were washedwith distilled water and streptavidin-HRPO diluted to approximately 200ng/mL was added to the plates and allowed to incubate for 30 minutes atroom temperature. The plates were washed with distilled water ando-phenylenediamine substrate was used as the chromagen to generatesignal and the optical density at 492 nm was measured.

Table 6 summarizes the assay signal (OD492 nm) for each antibody paircombination using 25 ng/ml of core1-169 antigen, which indicates whetheror not each binding pair is capable of forming a sandwich. An OD492value of at least 3× greater than the value generated by a negativecontrol (NC) monoclonal antibody as a capture or conjugate reagent areconsidered positive for core antigen detection.

TABLE 6 208A- 208A- 208A- 208A- 208A- 208B- 110 692 293 557 207-271 741(aa (aa (aa (aa (aa (aa 134-154 141-161) 134-154) 141-161) 141-161)134-154) & 141-161) 208A-110-Bt 0.18 0.14 0.20 0.17 0.17 0.20208A-692-Bt 0.54 0.35 0.51 0.53 0.39 0.44 208A-293-Bt 0.52 0.34 0.490.52 0.39 0.50 208A-557-Bt 0.44 0.13 0.41 0.32 0.18 0.38 208A-207-271-Bt0.38 0.42 0.42 0.82 0.36 0.30 208B-741-Bt 0.42 0.32 0.42 0.40 0.33 0.36208B-1096-Bt 0.19 0.15 0.24 0.17 0.18 0.23 208B-395-334-Bt 0.21 0.150.26 0.21 0.20 0.21 208B-612-226-Bt 0.26 0.18 0.28 0.23 0.24 0.23 NCmAb-Bt 0.16 0.15 0.23 0.18 0.17 0.18 C11-7-Bt 1.78 0.18 1.55 0.64 0.221.76 C11-9-Bt 2.59 0.58 1.84 1.18 0.44 2.32 208B- 208B- 208B- 1096395-334 612-226 NC C11-7 C11-9 (aa (aa (aa 134-154 capture (aa 115- (aa29- 151-171) 151-171) & 141-161) mAb 121) 37) 208A-110-Bt 0.21 0.22 0.200.15 0.18 0.13 208A-692-Bt 0.38 0.44 0.37 0.40 2.92 2.66 208A-293-Bt0.41 0.44 0.37 0.37 2.87 2.60 208A-557-Bt 0.35 0.28 0.25 0.18 0.22 0.14208A-207-271-Bt 0.33 0.37 0.33 0.37 2.47 2.20 208B-741-Bt 0.52 0.34 0.290.29 2.36 2.13 208B-1096-Bt 0.26 0.20 0.24 0.17 1.06 0.33208B-395-334-Bt 0.23 0.23 0.26 0.20 0.89 0.32 208B-612-226-Bt 0.70 0.830.23 0.21 1.52 0.64 NC mAb-Bt 0.21 0.22 0.22 0.18 0.17 0.14 C11-7-Bt0.45 0.46 0.53 0.22 0.23 1.04 C11-9-Bt 0.82 0.95 0.63 0.62 1.91 2.75

Example 13

Sequences of Anti-Core 134-171 Variable Domains.

A subset of the anti-HCV core 134-171 hybridomas were selected fordetermination of variable heavy (VH) and variable light (VL) chainnucleotide and deduced amino acid sequences. Total RNA was extractedfrom the hybridoma cells using Trizol (Invitrogen) or Tri-Reagent(Sigma) according to the manufacturer's recommendations. The heavy chainand light chain cDNA was generated from the extracted total RNA usingSuperscript III (Life Technologies) and oligo dT primers followingstandard protocols. The 5′ RACE (rapid amplification of cDNA ends)protocol was used to amplify the variable heavy and light chain cDNAsequences using a dC anchor primer(5′-AAGCAGTGGTATCAACGCAGAGTACCCCCCCCCCCCCCCCC-3′; SEQ ID NO:581) and ageneric primer specific to the constant region of the mouse heavy orlight chain (Novogen). Amplicons were cloned into a commerciallyavailable vector (pCR2.1-TOPO cloning kit, Invitrogen) per themanufacturer's directions and transformed into TOP 10 E. coli. At leasteight colonies were selected for PCR amplification of cloned variabledomain sequences using M13 forward and reverse primers. Amplicons weretreated with ExoSap (Affymetrix) prior to sequencing using M13 forwardprimer and BigDye Terminator v3.1 cycle sequencing kit (AppliedBiosystems, Foster City, Calif.). Sequences were obtained usingABI3130×1 automated sequencer and assembled and analyzed using VectorNTI software (Invitrogen).

Deduced amino acid sequences were aligned using ClustalW (Higgins etal., Nucleic Acids Res. 22:4673-4680, 1994) as implemented in the MEGASsoftware package (Tamura et al., Molecular Biology and Evolution 28:2731-2739, 2011). MEGAS software was used to determine groupings orclusters of related heavy chain amino acid sequences from the alignmentsand phylogenetic tree construction using the Neighbor Joining methodwith complete deletion of sequence gaps in the alignment. Tree topology,and hence clusters or groups therein, was examined for reliability byusing a bootstrap test from 1000 replicates. As a general rule, if thebootstrap value for a given interior branch is 95% or higher, then thetopology at that branch is considered “correct” (Nei and Kumar,Molecular Evolution and Phylogenetics, 2000; Oxford University Press,New York). Analysis of heavy chain variable domain sequences from 52anti-HCV core 134-171 monoclonals revealed the existence of 4 maingroups with bootstrap values >95%. Antibodies comprising three of thesegroups exhibited specificity for binding to one of each of the peptidesused for screening, i.e. Group B with peptide 1 (134-154), Group A withpeptide 2 (141-161), and Group C with peptide 3 (151-171). FIG. 2provides two representations of the tree topology.

Example 14 Generating the Neo4 Antibody

Six peptides from the HCV core region (genotype 1b) were synthesized.The HCV core amino acid (aa) positions covered by each peptide are asfollows: peptide 1, 98-110; peptide 2, 104-113; peptide 3, 104-121;peptide 4, 110-124; peptide 5, 101-112, and peptide 6, 153-165. Balb/Cmice were each immunized with a combination of 2 different peptides;peptides 1 with 6; peptides 2 with 3; peptides 4 with 5; and peptides 1with 5. All of the peptides were conjugated to keyhole limpet hemocyanin(KLH) at their C-terminus through an added cysteine residue. Mice wereboosted four times. Spleens were collected for 2 independent fusionsusing the PEG 1500 chemical method. The Myeloma fusion partner used wasSP2/0. Subcloning began after fusion screening. 150 cells were platedonto a 96 well plate which usually nets about 1 cell per well. Threerounds of subcloning were performed. Media used for the growth of cellsfrom stable clones was Iscove's Modified Dulbecco's Medium (IMDM) with10% FBS. Supernatants were analyzed by ELISA using the above corepeptides. Hybridoma supernatants and ascites generated fromELISA-positive clones were then screened by western blot for theirability to detect minicores. One hybridoma clone (Neo4) was identifiedthat gave an exceptionally strong signal at detecting minicore and p21core proteins. The isotype of this antibody was determined to be IgG1.Antibodies were subsequently purified with Protein G sepharose and usedfor the detection of minicores.

Study Population.

Hepatologists and other providers at the Icahn School of Medicine atMount Sinai recruited patients with a HCV viral load of 10 million IU/mLor above. Written informed consent was obtained and up to 16 mL of bloodwere collected, and serum was prepared. Data on age, sex, HCV genotype,viral load, and other medical conditions were extracted from medicalrecords. The study was conducted in compliance with the Icahn School

Blood was obtained from four patients with high-titer HCV and twonon-infected volunteers (Table 7). All four HCV-infected patients weremale and had a HCV viral load over 10 million IU/mL. Patients with ahigh HCV viral load were selected for this study because they wereexpected to have relatively high blood levels of HCV proteins, enablingthe development of methods for detection.

TABLE 7 Clinical characteristics of patients HCV Viral Patient Load HCVAge ID (IU/mL) Genotype (years) Sex Other P1 7.46 × 10⁷ 1b 49 M HIVco-infected P2 2.91 × 10⁷ 1b 63 M 5 years post kidney transplant P3 1.23× 10⁷ 1a 67 M — P4 >1 × 10⁸ * 1a 60 M 1 year post liver transplant and Bcell lymphoma N1 — — 55 M Healthy control N2 — — 65 F Healthy control *Value was above the upper limit of detection of the assay

Isolation and Characterization of Minicores and p21 Core in Blood.

The general procedure for isolating minicore and mature p21 core proteinfrom patient blood is shown in FIG. 4. An equal volume of 50 mM Tris (pH7.3) and 150 mM NaCl was added to 3.5 mL of serum and passed through a0.45 μm syringe filter to remove aggregates. ApoB-associatedlipoproteins were then precipitated by mixing an equal volume ofheparin/Mn⁺² solution containing 60 mM Tris (pH 7.3), 110 mM MnCl₂.4H₂O,154 mM NaCl, and 400 USP/mL heparin. Solution was incubated for 1 h onice in the dark. The precipitate was recovered by centrifugation at3,000×g (30 min; 4° C.) and washed with gentle resuspension three timeswith 3.5 mL of an ice-cold solution containing 50 mM Tris (pH 7.3), 55mM MnCl₂.4H₂O, 154 mM NaCl, and 200 USP units/mL heparin. Heparin/Mn⁺²was then removed: Manganese was removed from the heparin/Mn² pellet byresuspension in 2.0 or 2.5 mL of 10% NaHCO₃, which precipitates aninsoluble Mn(HCO₃)₂ that is removed by centrifugation at 1,500×g (15min; 4° C.). Heparin was removed by dialyzing 3 times (in 24 h) with oneliter of 5% BaCl₂ in 20 mM Tris (pH 7) in a Slide-A-Lyzer Cassette G2(Thermo Scientific) with a 20,000 MWCO. The supernatant was collectedfrom the dialysis unit and the heparin-BaCl₂ insoluble complex wasremoved by centrifugation at 1,500×g (15 min; 4° C.). Excess BaCl2 wasremoved from the supernatant by dialyzing 3 times (in 24 h; 4° C.) withone liter of THE buffer consisting of 20 mM Tris (pH 7.0), 0.15 M NaCland 1 mM EDTA. The supernatant was collected from the dialysis unit andstored at −80° C. After thawing, the supernatant was concentrated usinga 15 mL Amicon Ultra centrifugal filtration unit with a 100 kDa MWCO(Millipore) (3,200×g; 90 min; 4° C.). Concentrated volumes rangedbetween 130 μL and 210 μL. Finally, samples were delipidated to allowfor loading onto a Western blot gel. Samples were delipidated using anextraction method previously described for whole serum and plasma whichpartitions proteins into an aqueous phase and lipids into an organicphase using a mixture of butanol and diisopropyl ether (butanol:DIPE at40:60; v/v) (see, Cham et al., J Lipid Res 1976; 17:176-81, hereinincorporated by reference). The original protocol was modified by usingonly a 1× volume of the 40:60 butanol:DIPE mixture to our sample ratherthan a 2× volume. It was found that the 2× volume can greatly dehydratethe aqueous phase. Also, instead of extracting by end-over-end tuberotation of the samples for 30 minutes, the samples were just brieflyvortexed. The lipid-containing organic and protein-containing aqueousphases were separated by centrifugation at 400×g (2 min; 25° C.). Thelower aqueous phase was immediately collected, placed on ice for 1 h,and then stored at −80° C. Incubation of the protein-containing aqueousphase on ice for 1 h followed by freeze-thawing led to the formation ofa precipitate that was pelleted by gentle centrifugation at 1,000×g (30sec; 4° C.). The pellet was solubilized in a solution containing 8 MUrea and 1% SDS, sonicated, adjusted with NuPage sample buffer, heatedand loaded onto NuPAGE gels. This pellet contained core and minicoreproteins. These proteins were not detected in the supernatant. Afterelectrophoresis, gels were processed for western analysis. Proteaseinhibitors, Complete-EDTA free (Roche) and 1 mM phenylmethylsulfonylfluoride (PMSF) were used throughout the purification procedure.

HCV-Infected Cell Culture Lysate.

Huh-7.5 cells infected with a Con1/JFH chimeric HCV were were lyseddirectly in culture dishes after three washes with Dulbecco's phosphatebuffered saline with 2×LDS NuPage sample buffer (Life Technologies)containing 4% lithium dodecyl sulfate and 10% 2-mercaptoethanol.

Western Blot Analysis.

Samples were electrophoresed in 10% NuPAGE Bis-Tris gels (LifeTechnologies) and the proteins were transferred to 0.2 uM pore size,polyvinylidene difluoride (PVDF) membranes. Antibodies targeting theC-terminal portion of p21 core and used to detect minicores were acombination of Neo4 monoclonal antibody at a concentration of 2 ug/mlmixed with mAb1 (Cll-3) at 1 ug/ml, that we previously described (see,Eng et al., J Virol 2009; 83:3104-14, herein incorporated by reference).Antibody targeting the N-terminal portion of core is C11-10 (AbbottLabs, epitope 32-36).

Quantification of HCV RNA.

QIAamp Viral RNA mini kit (Qiagen) was used to purify RNA from serum,Hep/Mn⁺² pellet and supernatant. For the heparin/Mn⁺² pellet andsupernatatant, heparin was removed from the purified RNAs by treatingwith heparinase I (Sigma) (Johnson et al., Biotechniques 2003;35:1140-2, 1144, herein incorporated by reference). Reversetranscription (RT) of RNA was performed using SuperScript IIIFirst-Strand Synthesis (Invitrogen) and random hexamers. RT-reactionproducts were then used for quantitative-PCR (q-PCR) using theLightCycler 480 SYBR Green I Master kit and the LightCycler 480instrument (Roche).

Minicores were present in the blood of all four patients (FIG. 6). Therelative amounts of p21 core, 70 minicore and 91 minicore varied frompatient to patient. Patient samples P1, P2, and P4 had prominent p21core bands. Interestingly, P1, P2, and P4 were immune compromised as aresult of HIV infection or immunosuppressive drugs, while P3 had noknown immunological deficiency (Table 7). P1, P2, and P3 had prominent70 minicore bands, and P4 had a prominent 91 minicore band. A cellculture lysate of HCV-infected Huh-7.5 cells provided molecular weightmarkers of the core protein isoforms (FIGS. 6 and 7, lanes C). Toconfirm the identity of the core isoform bands, duplicate Western blotsof sample P4 were probed with antibodies specific for either the N- orthe C-terminal portion of the core protein (FIG. 7), allowing detectionof p21 core only (N-term) or p21 plus the 70 and 91 minicores (C-term).

This study provides direct evidence that HCV-expressed non-classicalproteins (minicores) are present in blood during natural infections inaddition to the conventional proteins. Minicores were detected in allfour high HCV viral load patients. The presence of relatively largequantities of minicores in blood suggests that they enhance viraltransmission and/or pathogenesis/persistence.

Example 15 Neo4 Antibody Sequencing

This example describes how the nucleotide and deduced amino acidsequences of the light and heavy chain variable domains of the Neo4monoclonal antibody were determined. Total cellular RNA was extractedfrom the anti-HCV core Neo4 hybridoma cells using Trizol (Tri-ZOL,Invitrogen) per manufacturer's recommendations. To sequence the variabledomains, cDNA was generated from the extracted RNA using Superscript III(Life Technologies) and an oligo-dT primer (Novagen). Amplification ofthe respective immunoglobulin variable sequences was performed by 5′RACEPCR using a dC-anchor primer and a murine consensus heavy or light chainprimer. Purified products were cloned into pCR™2.1-TOPO® (LifeTechnologies) which was then used to transform E. coli. For sequenceanalysis, variable heavy and variable light chain fragments frommultiple E. coli colonies were PCR amplified using M13 forward andreverse primers. Sequencing was performed using the BigDye Terminatorv3.1 cycle sequencing kit (Applied Biosystems) on the ABI 3130×1 GeneticAnalyzer. At least 5 heavy or light chain clones were viewed, analyzed,and annotated using Vector NTI Advance (Life Technologies) to create theconsensus nucleotide sequences. FIG. 8 shows the amino acid sequence ofthe light chain variable region (FIG. 8A, SEQ ID NO:583) and the heavychain variable region (FIG. 8B, SEQ ID NO:584) of Neo4. The CDRs wereidentified by visual inspection of the deduced amino acid sequencesrelative to the locations of conserved features within the respectivedomains (see, Kabat et al., (1987) Sequences of Proteins ofImmunological Interest, 4th ed., U. S. Govt. Printing Office No.165-492, Bethesda, Md.). FIG. 8A shows the light chain CDRs underlined,which include CDRL1 (RASKSVNEYGYTYMH; SEQ ID NO:585), CDRL2 (LASNLDS;SEQ ID NO:586), and CDRL3 (QHSRELPYT, SEQ ID NO:587). FIG. 8B shows theheavy chain CDRs underlined, which include CDRH1 (GFSITSSVYCWQ; SEQ IDNO:588), CDRH2 (RICYDGSVDYSPSITS; SEQ ID NO:589), and CDRH3(ENHIDYYDTTYPSFDV; SEQ ID NO:590). FIG. 9A shows the nucleic acidsequence encoding the light chain variable region of Neo4 (SEQ IDNO:591), and FIG. 9B shows the nucleic acid sequence encoding the heavychain variable region of Neo4 (SEQ ID NO:592).

Example 16 Neo4 and CLL-3 (ABT-4) Antibody Epitope Mapping

Purified Neo4 and C11-3 (ABT-4) monoclonal IgG samples were analyzed forreactivity to overlapping HCV core antigen peptides by EIA.Biotin-(SGSG)-15 mer HCV core-amide peptides were synthesized thatoverlapped by two amino acids and encompassed the entire length of HCVcore protein amino acids 1-191 (Mimotopes). Each of the 89 synthesizedpeptides was coated in an individual micro-titer well as indicated onthe peptide plate key (Table 8) as shown. The coated plates were keptdry in a sealed foil pouch and stored at <10 degrees C. until ready foruse. A peptide coated plate for each test antibody was blocked using 3%BSA in PBS with 0.5% Tween 20. The blocked plates were washed withdistilled water and a 2 ug/mL solution of purified IgG from each testsample diluted in BSA block solution was added to each peptide coatedwell of the blocked plate and allowed to incubate at room temperaturefor at least one hour. Following incubation the plates were washed withdistilled water. A 200 ng/mL solution of peroxidase labeled F(ab′)2fragment goat anti-mouse IgG Fc fragment specific (JacksonImmunoresearch) in BSA block solution was added to all wells andincubated at room temperature for 30 minutes. The plates were washedwith distilled water and o-phenylenediamine substrate was used as thechromagen to generate signal. Plates were read at 492 nm and the resultswere analyzed. Wells were considered positive if they had an EIA signalat least 3 times greater than background. The reactivity of ABT-4 to thepeptides is shown with underlining in Table 9, while the reactivity ofthe Neo4 antibody to the peptides is shown with underlining in Table 10.As shown in these tables, the ABT-4 mAb reacts with peptide numbers48-53 while the Neo4 reacts with peptide numbers 50-55.

TABLE 8 Peptide Plate Key 1 2 3 4 5 6 7 8 9 10 11 12 A 1 9 17 25 33 4149 57 65 73 81 89 B 2 10 18 26 34 42 50 58 66 74 82 Blank C 3 11 19 2735 43 51 59 67 75 83 Blank D 4 12 20 28 36 44 52 60 68 76 84 Blank E 513 21 29 37 45 53 61 69 77 85 Blank F 6 14 22 30 38 46 54 62 70 78 86Blank G 7 15 23 31 39 47 55 63 71 79 87 Blank H 8 16 24 32 40 48 56 6472 80 88 Blank

TABLE 9 HCV ABT-4 1 2 3 4 5 6 7 8 9 10 11 12 A 0.05 0.06 0.07 0.06 0.070.07 0.97 0.07 0.07 0.07 0.07 0.07 B 0.06 0.06 0.06 0.06 0.06 0.07 0.970.07 0.07 0.07 0.07 0.07 C 0.06 0.07 0.06 0.06 0.06 0.06 1.00 0.07 0.070.07 0.07 0.07 D 0.06 0.07 0.06 0.06 0.06 0.07 0.96 0.07 0.06 0.06 0.060.06 E 0.05 0.06 0.06 0.06 0.06 0.07 0.36 0.06 0.06 0.06 0.06 0.06 F0.07 0.06 0.06 0.06 0.06 0.07 0.08 0.06 0.06 0.06 0.06 0.06 G 0.06 0.060.06 0.06 0.06 0.07 0.06 0.06 0.06 0.06 0.07 0.06 H 0.06 0.06 0.07 0.070.08 0.81 0.07 0.07 0.08 0.07 0.09 0.07

TABLE 10 HCV Neo4 1 2 3 4 6 6 7 8 9 10 11 12 A 0.06 0.05 0.04 0.04 0.050.04 0.05 0.05 0.04 0.04 0.04 0.05 B 0.06 0.05 0.05 0.04 0.04 0.04 1.030.05 0.05 0.05 0.04 0.05 C 0.05 0.05 0.05 0.04 0.04 0.05 0.96 0.05 0.050.10 0.05 0.05 D 0.06 0.07 0.05 0.04 0.05 0.05 0.93 0.06 0.04 0.05 0.050.05 E 0.05 0.05 0.05 0.04 0.04 0.04 0.96 0.05 0.05 0.05 0.05 0.04 F0.06 0.06 0.07 0.05 0.05 0.06 1.01 0.05 0.04 0.06 0.05 0.05 G 0.07 0.060.05 0.05 0.05 0.05 0.97 0.04 0.05 0.05 0.05 0.04 H 0.06 0.06 0.05 0.060.05 0.04 0.04 0.04 0.04 0.05 0.05 0.05

A summary of the peptide reactivity of ABT-4 (C11-3) and Neo4 isprovided in Table 11 below.

TABLE 11 Peptide Reactivity Summary HCV Core monoclonal HCV antibodyCore SEQ ABT-4 Neo4 Peptide HCV ID HCV OD OD # Core Peptide Sequence NOCore Region (A492nm) (A492nm) 48 SGSGGWLLSPRGSRPSWGP 593 aa 95-109 0.950.04 49 SGSGLLSPRGSRPSWGPTD 594 aa 97-111 1.03 0.05 50SGSGSPRGSRPSWGPTDPR 595 aa 99-113 1.03 1.03 51 SGSGRGSRPSWGPTDPRRR 596aa 101-115 1.01 0.96 52 SGSGSRPSWGPTDPRRRSR 597 aa 103-117 1.01 0.93 53SGSGPSWGPTDPRRRSRNL 598 aa 105-119 0.36 0.96 54 SGSGWGPTDPRRRSRNLGK 599aa 107-121 0.08 1.01 55 SGSGPTDPRRRSRNLGKVI 600 aa 109-123 0.08 0.97

Shown in Table 12 below, are the reactive HCV core peptides aligned,with the minimal overlap underlined. For Abt-4, the reactive sequence isshown to span from amino acid 95 to amino acid 117 of the core peptidesequence, with the minimum reactive sequence spanning from amino acid103 to amino acid 109. For Neo4, the reactive sequence is shown to spanfrom amino acid 99 to amino acid 123 of the core peptide sequence, withthe minimum reactive sequence spanning from amino acid 109 to amino acid113.

TABLE 12 Abt4 (C11-3) Neo4 GWLLSPRGSRPSWGP (SEQ ID NO: 601)SPRGSRPSWGPTDPR (SEQ ID NO: 606)   LLSPRGSRPSWGPTD (SEQ ID NO: 602)  RGSRPSWGPTDPRRR (SEQ ID NO: 607)     SPRGSRPSWGPTDPR (SEQ ID NO: 603)    SRPSWGPTDPRRRSR (SEQ ID NO: 608)      RGSRPSWGPTDPRRR (SEQ ID NO: 604)      PSWGPTDPRRRSRNL (SEQ ID NO: 609)        SRPSWGPTDPRRRSR (SEQ ID: 605)        WGPTDPRRRSRNLGK (SEQ ID NO: 610)          PTDPRRRSRNLGKVI (SEQ ID: 611)

Example 17 HCV Core Peptide Reactivity of Anti-Core MonoclonalAntibodies

Assay wells were coated with sheep anti-mouse IgG Fc specific antibodyand incubated overnight. The coating solution was then removed; thewells blocked using BSA/tween in PBS and then washed with dH2O. Seriallydiluted antibody (in block) test samples (Neo4 antibody and Abt-4antibody) were then added, the plates incubated for at least 1 hour andthen washed. Next, biotin labeled peptides (diluted to 500 ng/mL inblock) were added, the plates incubated for 10 minutes while shaking at700 rpm and then washed. Peroxidase conjugated streptavidin (diluted to200 ng/mL in block) was then added to all assay wells, incubated for 20minutes while shaking at 700 rpm and then washed. Finally, color wasdeveloped using OPD and signal quenched using 1N H2SO4. Signal was readat 492 nm. Data are tabulated and summarized in Table 13 below.

TABLE 13 normal mouse serum sera dil (1:X) bt-pep 500 1,000 2,000 4,0008,000 16,000 32,000 64,000 128,000 256,000 512,000 1.1 MM WT1STHV 0.06250.0552 0.0511 0.053  0.0537 0.06  0.0635 0.0628 0.067  0.0675 0.07430.0585 Mut1T110N 0.2575 0.0516 0.0617 0.0501 0.057  0.0626 0.0667 0.07020.0651 0.0677 0.0759 0.0631 Mut6T110S 0.2025 0.0545 0.0531 0.0505 0.05230.0611 0.0591 0.0631 0.069  0.0674 0.065  0.0621 C113WT 0.0603 0.056 0.0534 0.0532 0.0589 0.0537 0.0644 0.0623 0.066  0.066  0.0708 0.0586C113T109N 0.0619 0.051  0.0534 0.0536 0.0607 0.0714 0.0751 0.0674 0.068 0.0629 0.0931 0.0726 92-133 0.0636 0.0521 0.0603 0.0533 0.0756 0.10090.0822 0.0656 0.0668 0.0615 0.0715 0.0691 blank 0.0563 0.0488 0.05380.0486 0.0508 0.0549 0.0679 0.0596 0.0631 0.065  0.0772 0.0783 blank0.0691 0.0503 0.048  0.0494 0.0543 0.0534 0.0694 0.0584 0.0622 0.06540.0637 0.0772 HCV C11-3 ng/mL Ab bt-pep 2000 1000 500 250 125 62.5 31.2515.625 7.813 3.906 1.953 0.977 WT1STHV 2.1334 2.0583 2.1436 1.86622.038  2.098  1.8905 1.5079 1.1164 0.6026 0.3531 0.1881 Mut1T110N 0.09150.0721 0.0765 0.0857 0.1268 0.1549 0.1066 0.1103 0.0966 0.079  0.08540.0755 Mut6T110S 1.7329 2.1499 2.237  1.8305 2.0224 2.1105 1.5367 0.80750.3435 0.2271 0.0964 0.1005 C113WT 1.821  2.3479 2.3525 1.8679 2.37152.1987 1.758  1.0506 0.6131 0.2717 0.1721 0.1029 C113T109N 0.0707 0.08060.0882 0.0963 0.1475 0.2015 0.1599 0.1161 0.1192 0.0833 0.1143 0.104192-133 1.4473 1.8692 1.9517 1.6777 1.8343 1.9204 1.5476 1.0138 0.56230.1832 0.1342 0.1163 blank 0.0657 0.071  0.0995 0.0639 0.0736 0.09870.1178 0.1047 0.101  0.0831 0.0937 0.0959 blank 0.0743 0.0711 0.06550.0644 0.0791 0.1032 0.1452 0.0979 0.1179 0.0856 0.0763 0.142 HCV Neo4ng/mL Ab bt-pep 2000 1000 500 250 125 62.5 31.25 15.625 7.813 3.9061.953 0.977 WT1STHV 1.8978 2.0033 1.9126 1.9886 1.9279 1.8543 1.67011.4209 1.0414 0.6691 0.4092 0.2204 Mut1T110N 0.0882 0.0678 0.065  0.089 0.2042 0.1391 0.0938 0.0771 0.0729 0.0719 0.0728 0.0619 Mut6T110S 1.16671.165  1.1716 1.1549 1.2728 1.1072 0.4805 0.2502 0.1316 0.0847 0.07170.0644 C113WT 0.0766 0.2219 0.0561 0.0607 0.0599 0.0626 0.0622 0.06970.0731 0.0694 0.0765 0.0584 C113T109N 0.059  0.0591 0.0803 0.1105 0.08580.1106 0.0815 0.0736 0.0751 0.0717 0.0853 0.0784 92-133 1.3067 1.41381.4754 1.4019 1.3064 1.4726 1.1055 0.8058 0.45  0.2166 0.1293 0.0978blank 0.0643 0.0581 0.0663 0.0538 0.0534 0.0641 0.0689 0.0674 0.06960.0728 0.093  0.0715 blank 0.0676 0.0515 0.051  0.0526 0.0512 0.06420.0723 0.0687 0.0708 0.0687 0.0715 0.0767

Sequences of HCV core peptides are shown below where # denotes position110 of the HCV core protein sequence.

          # WT1STHV GSRPSWGPTDPRHRSRNVGKVID (SEQ ID NO: 612) MUT1T110N  

(SEQ ID NO: 613) MUT6T110S   

(SEQ ID NO: 614) C113WT RGSRPSWGPTD (SEQ ID NO: 615) C113T110N  

(SEQ ID NO: 616)

Differences in sequence relative to genotype 1 at position 110 arehighlighted. Reactivity of C11-3 (Abt4) and Neo4 at the 15.6 ng/mLdilution are summarized below in Table 14.

TABLE 14 HCV HCV C11-3 Neo4 bt-pep 15.6 ng/mL 15.6 ng/mL WT1STHV 1.50791.4209 Mut1T110N 0.1103 0.0771 Mut6T110S 0.8075 0.2502 C113WT 1.05060.0697 C113T109N 0.1161 0.0736 92-133 1.0138 0.8058 blank 0.1047 0.0674blank 0.0979 0.0687

Both Neo4 and C11-3 (Abt-4) are sensitive to the T110N mutation commonamong non-genotype 1 and 2 isolates. However, Neo4 appears to moresusceptible to the T110S mutation compared to Abt4. Neo4 does not bindto peptides terminating at position 111 indicating that the Neo4 epitopeinvolves HCV core sequences downstream of position 111. This isconsistent with the epitope mapping using the core peptide librarywherein the Neo4 and Abt4 epitopes overlap but the Neo4 epitope isshifted downstream (C-terminally) relative to Abt 4 by about 3-4 aminoacids.

Example 18 Monoclonal Antibodies Neo4 and 750 Used in CombinationEnhance the Detection of HCV Core and 91-Minicore Proteins

Core and 91-minicore proteins were expressed in 293T cells bytransfecting plasmids which encode the respective proteins. The core andminicore plasmid constructs also contained the 5′ two-thirds of the HCVE1 gene which normally follows the core gene in the HCV genome. Coreprotein sequences are cleaved away from E1 protein by cellular signalpeptidase and signal peptide peptidase to yield the mature core orminicore proteins. The 91-minicore construct begins with a start codonat codon 91 of the core gene. HCV sequences in both constructs werecodon-optimized for enhanced expression. Cell extracts were prepared 48hours post-transfection using a 2× lithium dodecyl sulfate (LDS) gelloading buffer followed by sonication. Extracts were diluted with 1×LDSloading buffer and were run on 10% Bis-Tris NuPage gels. Proteins weretransferred to 0.2 uM pore size polyvinylidene difluoride (PVDF)membranes for Western blot analysis. The HCV core epitopes targeted byNeo4 and 208A-750 monoclonal antibodies (aa98-110 and aa141-161,respectively as immunogens) are downstream of amino acid 91 and are thuscapable of detecting both core and 91-minicore. The concentration ofeach antibody used in the Western blot analysis was at 2 μg/ml.

As shown in FIG. 10, when antibodies Neo4 and 208B-750 are used incombination, the signal intensity for core and 91-minicore are enhancedas compared to when the antibodies are used individually.

APPENDIX A DESCRIPTION OF SEQUENCES mAb SEQ SEQ mAb Clone IDNucleic Acid Seq ID Clone Amino Acid Seq 208A- 1GAGGTCCAGCTGCAACAGTCTGGACCTGAGTT 2 208A-1064H EVQLQQSGPELVKPGASVKISCK1064H GGTGAAGCCTGGGGCCTCAGTGAAGATATCTT TSGYTFTEYAMHWMKQSHGKSLEGCAAGACTTCTGGATACACTTTCACTGAATAC WIGGINPTNGDTIYNQHFKDKAKGCCATGCACTGGATGAAGCAGAGCCATGGAAA LTVDRSSSTAYMELRSLTSDDSAGAGCCTTGAGTGGATTGGAGGTATCAATCCTA LFYCARRELDYFASWGQGTTLTVCTAATGGTGATACAATCTACAACCAGAAGTTC SS AAGGACAAGGCCAAATTGACTGTAGACAGGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCC TGACATCTGACGATTCTGCATTATTTTATTGTGCAAGACGGGAACTGGACTACTTTGCCTCCTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B- 3GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 4 208B-1094H EVQLQQSGPELVKPGASVKISCK1094H GGTGAAGCCTGGGGCCTCAGTGAAGATATCCT TSGYTFTEYAMHWMKQSHGKSLEGCAAGACTTCTGGATACACTTTCACTGAATAC WIGGINPTNGDTIYNQRFKDKAKGCCATGCACTGGATGAAGCAGAGCCATGGAAA LTVDRSSSTAYMELRSLTSDDSAGAGCCTTGAGTGGATTGGCGGTATCAATCCTA LFYCARRELDYFASWGQGTTLTVCTAATGGTGATACAATCTACAACCAGAGGTTC SS AAGGACAAGGCCAAATTGACTGTAGACAGGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCC TGACATCTGACGATTCTGCATTATTTTATTGTGCAAGACGGGAACTGGACTACTTTGCCTCCTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA208A-293H 5 GAGGTCCAGCTGCAACAGTCTGGACCTGAACT 6 208A-293HEVQLQQSGPELVKPGASVKISCK GGTGAAGCCTGGGGCCTCAGTGAAGATATCCTASGFTFTEYAMHWMKQSHGKSLE GTAAGGCTTCGGGATTCACTTTCACTGAATACWIGGINPTNGDAIYNQHFKDKAK GCCATGCACTGGATGAAACAGAGCCATGGAAALTVDRSSSTAYMELRSLTSDDSA GAGCCTTGAGTGGATTGGAGGTATCAATCCTALFYCARRELDYFPSWGQGTTLTV CTAACGGTGATGCAATCTACAACCAGAAGTTC SSAAGGACAAGGCCAAGTTGACTGTAGACAGGTC CTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGACGATTCTGCATTATTTTATTGT GCAAGACGGGAACTGGACTACTTTCCCTCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 208A-222H 7GAGGTCCAGCTGCAACAGTCTGGACCTGAACT 8 208A-222H EVQLQQSGPELEKPGASVRISCKGGAAAAGCCTGGGGCTTCAGTGAGGATATCCT TSGYTFTEYAMHWVKQSHGKSLEGCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPNNGNAIYNQIFKDKATGCCATGCACTGGGTGAAGCAGAGCCATGGAAA LTVDRSSSTAYMGLRSLTFGDSGGAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDYFDYWGQGASLTVACAATGGCAATGCTATCTACAACCAGATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTCCTCCAGCACAGCCTACATGGGCCTCCGCAGCC TGACATTCGGGGATTCTGGAGTCTACTTCTGTGTAAGACGACAACTGGACTACTTTGACTATTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA208A-605H 9 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 10 208A-605HEVQLQQSGPELEKPGASVKISCK GGAAAAGCCTGGGGCTTCAGTGAAGATATCCTTSGYTFTEYAIHWVKQSHGMSLE GCAAGACTTCTGGATACACATTCACTGAATACWIGGINPSNGNAIYNQIFKDKAT GCCATACACTGGGTGAAGCAGAGCCATGGAATLTVDRSSSTAYMGLRSLTFGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTAVYFCVRRQLDFFDYWGQGASLTV GCAATGGCAATGCTATCTACAACCAAATATTC SSAAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAGCACAGCCTACATGGGCCTCCGCAGCCTGACATTTGGGGATTCTGGAGTCTACTTCTGT GTAAGACGACAACTGGACTTCTTTGACTATTGGGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208B-560H 11GAGGTCCAGCTGCAACAGTCTGGACCTGAGCT 12 208B-560H EVQLQQSGPELEKPGASVKISCKGGAAAAGCCTGGGGCTTCAGTGAAGATATCCT TSGYTFTEYAMHWVKQSHGMSLEGCAAGACTTCTGGATACACATTCACTGAATAC WIGGINPSNGNAIYNQIFKDKATGCCATGCACTGGGTGAAGCAGAGCCATGGAAT LTVDRSSSTAYMGLRSLTFGDSGGAGCCTTGAGTGGATTGGAGGTATTAATCCTA VYFCVRRQLDFFDYWGQGASLTVGCAATGGCAATGCTATCTACAACCAGATATTC SS AAGGACAAGGCCACACTGACTGTGGACAGGTCCTCCAGCACAGCCTACATGGGCCTCCGCAGCC TGACATTTGGGGATTCTGGAGTCTACTTCTGTGTAAGACGACAACTGGACTTCTTTGACTATTG GGGCCAGGGCGCCTCTCTCACAGTCTCCTCA208A-830H 13 GAGGTCCGGCTGCAGCAGCCTGGACCTGAGGT 14 208A-830HEVRLQQPGPEVEKPGASVKISCK GGAAAAGCCTGGGGCTTCAGTGAAGATATCCTTSGYTFTEYAIHWVEQSHGESLE GCAAGACTTCTGGATACACATTCACTGAATACWIGGINPSNGDPIYNQIFKDKAT GCCATCCACTGGGTGAAACAGAGCCATGGAGALTVDRSSNTAYMGLRSLTVGDSG GAGCCTTGAGTGGATTGGAGGTATTAATCCTAVYFCVRRQLDYFDFWGQGASLTV GCAATGGCGATCCTATCTATAACCAGATATTC SSAAGGACAAGGCCACACTGACTGTGGACAGGTC CTCCAACACAGCCTACATGGGCCTCCGCAGCCTGACAGTTGGGGATTCTGGAGTCTACTTCTGT GTTAGACGACAACTGGACTACTTTGACTTTTGGGGCCAGGGCGCCTCTCTCACAGTCTCCTCA 208A-134H 15CAGGGTCAGATGCAGCAGTCTGGAGCTGAACT 16 208A-134H QGQMQQSGAELAKPGASVKLSCKGGCGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYISWLEQFPGQSLEGCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQTATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQLSSLTTEDSAGAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVSGAACTGGTAATACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATCCTCCAGTACAGCCTACATGCAACTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGTGCGATAAGTGGGACGGGATTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAACA208A-692H 17 CAGGGTCAGATGCAGCAGTCTGGAGCTGAACT 18 208A-692HQGQMQQSGAELAKPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTTSGFTFSSSYISWLEQFPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGTWIAWIFAGTGNTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAAGCCTGGACALTVDTSSSTAYMQLSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTGIYYCAISGTGFTYWGQGTLVTVS GAACTGGTAATACTAACTATAATCAGAAGTTC AACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGTACAGCCTACATGCAACTCAGCAGCCTGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGATTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAACA 208A-557H 19CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 20 208A-557H QGQMQQSGAELVEPGASVKLSCKGGCGAAGCCTGGGGCTTCAGTGAAACTGTCCT TSGFTFSSSYISWLEQFPGQSLEGCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQTATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQLSSLTTEDSAGAGTCTTGAGTGGATTGCATGGATTTTTGCTG IYYCAISGTGFTYWGQGTLVTVSGAACTGGTAATACTAATTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATCCTCCAGTACAGCCTACATGCAACTCAGCAGCC TGACAACTGAGGACTCTGCCATCTATTACTGTGCGATAAGTGGGACGGGATTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA 208A-352H21 CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 22 208A-352H QGQMQQSGAELVEPGASVKLSCKGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSFISWLEQFPGQSLEGCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTNYNQKFTDKAQTTTATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTSSSTAYMQFSSLTTEDSAGAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVSGAACTGGAAATACTAACTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAATTCAGCAGCC TGACGACTGAGGACTCTGCCATCTATTACTGTGCGATAAGTGGGACGGGGTTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCA 208A-983H23 CAGGGTCAGATGCAGCAGTCTGGAGCTGAGCT 24 208A-983H QGQMQQSGAELVEPGASVKLSCKGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCT TSGFTFSSSYFSWLEQFPGQSLEGCAAGACTTCTGGCTTCACCTTCAGCAGTAGT WIAWIYAGTGNTIYNQKFTDKAQTATTTTAGTTGGTTGAAGCAAAAGCCTGGACA LTVDTASSTAFMQLSSLTIEDSAGAGTCTTGAGTGGATTGCATGGATTTATGCTG IYYCAISGTGFTYWGQGTLVTVSGAACTGGTAATACTATCTATAATCAGAAGTTC A ACAGACAAGGCCCAACTGACTGTAGACACAGCCTCCAGCACAGCCTTCATGCAACTCAGCAGCC TGACAATTGAGGACTCTGCCATCTACTACTGTGCGATAAGTGGGACGGGGTTTACTTACTGGGG CCAAGGGACTCTGGTCACTGTCTCTGCAACA208B-281H 25 CAGGGTCAGCTGCAGCAGTCTGGAGCTGAGCT 26 208B-281HQGQLQQSGAELVEPGASVKLSCK GGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTTSGFTFSSSYISWLEQRPGQSLE GCAAGACTTCTGGCTTCACCTTCAGCAGTAGTWIAWIYAGTGGTNYNQKFTDKAQ TATATAAGTTGGTTGAAGCAAAGGCCTGGACALTVDTSSSTAYMQFSSLTTEDSA GAGTCTTGAGTGGATTGCATGGATTTATGCTGIYYCAISGTGFIYWGQGTLVTVS GAACTGGTGGTACTAACTATAATCAGAAGTTC AACAGACAAGGCCCAACTGACTGTAGACACATC CTCCAGCACAGCCTACATGCAATTCAGCAGCCTGACAACTGAGGACTCTGCCATCTATTACTGT GCGATAAGTGGGACGGGGTTTATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 208B-471H 27CAGGGTCAGTTGCAGCAGTCTGGACCAGTACT 28 208B-471H QGQLQQSGPVLVKPGASEILYCKGGTGAAGCCTGGGGCTTCAGAAATACTATACT TSGFTESSTYISWLKQKPGQSLEGCAAGACTTCTGGCTTCACCTTCAGCAGTACC WIAWIYAGTGATNYNQKFTGKAQTATATAAGTTGGTTGAAGCAAAAGCCTGGACA LTVDASSNTAYMHFSGLTPEDSAGAGTCTTGAGTGGATTGCGTGGATTTATGCTG IYYCAISGAGVYWGQGTLVTVSAGAACTGGTGCTACTAATTATAATCAGAAGTTC ACAGGCAAGGCCCAACTGACTGTAGACGCTTCCTCCAACACAGCCTACATGCACTTCAGCGGCC TGACACCTGAGGACTCTGCCATCTATTACTGTGCAATTTCTGGGGCGGGGGTTTACTGGGGCCA AGGGACTCTGGTCACTGTCTCTGCA 208A-207H 29CAGGGCCAACTGCAGCAGCCTGGGGCTGAGTT 30 208A-207H QGQLQQPGAEFVKPGASLKLSCRTGTGAAGCCTGGGGCTTCACTGAAGCTGTCCT ASGYTFTSYWIHWVKQRPGQGLEGCAGGGCTTCTGGCTACACCTTCACCAGCTAC WIGEIDPSDSYINQNQHFRGKATTGGATACACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSSTAYMELSGLTSEDSAAGGCCTTGAGTGGATTGGAGAAATTGATCCTT VYYCARHYYGVLDSWGQGTTLTVCTGACAGTTATATTAACCAGAATCAAAAGTTC SS AGGGGCAAGGCCACATTGACTGTGGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCGGCC TGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAGACATTACTACGGTGTTCTTGACTCCTG GGGCCAAGGTACCACTCTCACAGTCTCCTCAA CA208A-638H 31 CAGGGCCAACTGCAGCAGCCTGGGGCTGAGTT 32 208A-638HQGQLQQPGAEFVKPGASLKLSCR TGTGAAGCCTGGGGCTTCACTGAAGCTGTCCTASGYTFTSYWIHWVKQRPGQGLE GCAGGGCTTCTGGCTACACCTTCACCAGCTACWIGEVDPSDSYINQNEKERGKAT TGGATTCACTGGGTGAAGCAGAGGCCTGGACALTVDKSSSTAYMQLGSLTSEDSA AGGCCTTGAGTGGATCGGAGAAGTTGATCCTTVYYCARHYYGVLDSWGQGTALTV CTGACAGTTATATTAACCAGAATGAAAAGTTC SSAGGGGCAAGGCCACATTGACTGTGGACAAATC CTCCAGCACAGCCTACATGCAGCTCGGCAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGT GCAAGACATTACTACGGTGTTCTTGACTCCTGGGGCCAAGGCACCGCTCTCACAGTCTCCTCA 208B-515H 33CAGGTCCAACTGCAGCAGCCTGGGGCTGAACT 34 208B-515H QVQLQQPGAELVKPGASLKLSCRTGTGAAGCCTGGGGCTTCACTGAAGCTGTCCT ASGYTFTSYWIHWVKQRPGQGLEGCAGGGCCTCTGGCTACACCTTCACCAGCTAC WIGEIDPSDSYTNYNQHFKGKATTGGATTCACTGGGTGAAGCAGAGGCCTGGACA LTVDKSSRAAYMQLSSLTSEDSAAGGCCTTGAGTGGATCGGAGAGATTGATCCTT VYYCARHYYGVEDSWGQGTHLTVCTGATAGTTATACTAACTACAATCAAAAGTTC SS AAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGGGCAGCCTACATGCAGCTCAGCAGCC TGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACATTACTACGGTGTCTTTGACTCCTG GGGCCAAGGCACCAAACTCACAGTCTCCTCA208A-874H 35 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCT 36 208A-874HQVQLQQPGAELVKPGASVKLSCK TGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTASGYTLSSYWMHWVKQRPGQGLE GCAAGGCTTCTGGCTACACCCTCAGTAGCTATWIGEIHPSDSYTSYNQHFKDKAT TGGATGCACTGGGTGAAGCAGAGGCCTGGACALTVDKSSSTAYMQLSSLTSEDSA AGGCCTTGAGTGGATCGGAGAGATTCATCCTTVYYCARGGYYRYDEFAYWGQGTL CTGATAGTTATACTAGCTACAATCAAAAGTTC VTVSAAAGGACAAGGCCACATTGACTGTAGACAAATC CTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGT GCAAGGGGGGGCTACTATAGGTACGACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTG TCTCTGCA 208B-911H 37CAGGTCCATCTGCAGCAGCCTGGGGCTGAGCT 38 208B-911H QVHLQQPGAELVRPGVSVKLSCKGGTGAGGCCTGGGGTTTCAGTGAAGCTGTCCT ASGYTETTYSINWMKQRPGQGLEGCAAGGCTTCTGGCTACACCTTCACCACCTAC WIGNIYPSTSHTNYNQHFRDKATTCGATAAACTGGATGAAGCAGAGGCCTGGACA MTVDKSSSTAYMQLSSPTSEDSAAGGCCTTGAGTGGATCGGAAATATTTATCCTT VYYCTINAYSMDYWGQGTSVTVSCTACCAGTCATACTAACTACAATCAAAAGTTC S AGGGACAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCC CGACATCTGAGGACTCTGCGGTCTATTATTGTACAATAAATGCCTATTCTATGGACTACTGGGG TCAAGGAACCTCAGTCACCGTCTCCTCA 208B- 39CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 40 208B-1096H QVQLQQSGAGLAKPGASVKMSCK1096H GGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWAKQRPGQGLEGCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPSSGYTEYNHEIQYKATAAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSTLTFEDSAGGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSAGCTCTGGTTATACTGAATACAATCATAAGATC CAGTACAAGGCCACTTTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAACTGAGCACCC TAACATTTGAAGACTCTGCAGTCTATTACTGTACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCAACA 208B-589H 41CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 42 208B-589H QVQLQQSGAGLAKPGASVKMSCKGGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWAKQRPGQGLEGCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPSSGYTEYNHEIQDKATAAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSSLTFEDSAGGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSAGCTCTGGTTATACTGAATACAATCATAAGATC CAGGACAAGGCCACATTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAACTGAGCAGCC TAACATTTGAAGACTCTGCAGTCTATTACTGTACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCA 208B-395H 43CAGGTCCAGCTTCAGCAGTCTGGGGCTGGACT 44 208B-395H QVQLQQSGAGLAKPGASVKMSCKGGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTANKMHWTHQRPGQGLEGCAAGGCTTCTGGCTATACCTTTACTGCCAAC WIGYIDPSSGYTQYNHEIQDKATAAGATGCACTGGACAAAACAGCGGCCTGGACA LTADTSSSTAYMQLSSLTFEDSAGGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSAGCTCTGGTTATACTCAATACAATCATAAGATC CAGGACAAGGCCACATTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAACTGAGCAGCC TAACATTTGAAGACTCTGCAGTCTATTACTGTACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCAGCA 208B-189H 45CAGGTCCACCTTCAGCAGTCTGGGGCTGAACT 46 208B-189H QVHLQQSGAELAKPGASVQMSCKGGCCAAACCTGGGGCCTCAGTGCAGATGTCCT ASGYTFTANKMHWARQRPRQGLEGCAAGGCTTCTGGCTACACCTTTACTGCCAAC WIGYIDPASGYTEYNQKIKDRATAAGATGCACTGGGCAAGACAGCGGCCTAGACA LTADESSSTAYMQLSSLTSEDSAGGGTCTGGAATGGATTGGATACATTGATCCTG VYYCTNFAYWGQGTLVTVSTCCTCTGGCTATACTGAATACAATCAGAAGATC AAGGACAGGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCC TGACATCTGAGGACTCTGCAGTCTATTACTGTACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTACAACA 208B-547H 47CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACT 48 208B-547H QVQLQQSGAELAKPGASVKMSCKGGCAAAACCTGGGGCCTCAGTGAAGATGTCCT ASGYTFTSNEMHWAKQRPGQGLEGCAAGGCTTCTGGCTACACCTTTACTAGCAAC WIGYIDPSSGYTEYNQKIEDKATAAGATGCACTGGGCAAAACAGCGGCCTGGACA LTADESSSTAYMQLSSLTSEDSAGGGTCTGGAATGGATTGGATACATTGATCCTA VYYCTNFAYWGQGTLVTVSAGCTCTGGTTATACTGAATACAATCAGAAGATC AAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCC TGACATCTGAGGACTCTGCAGTCTATTACTGTACAAATTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCAACA 208A-210H 49CAGGTCCAGTTGCAGCAGTCTGGACCTGAGTT 50 208A-210H QVQLQQSGPELVKPGASMRISCKGGTGAAGCCTGGGGCTTCAATGAGGATATCCT ASGYTFTSYYVHWIEQRPGQGLEGCAAGGCTTCTGGCTACACCTTCACAAGCTAC WIGCTYPGDVNTDYNEFFEGKATTATGTACACTGGATAAAGCAGAGGCCTGGACA LTADESSSTAYMQVSTLTSEDSAGGGACTTGAGTGGATTGGATGTATTTATCCTG IYFCVLYYYGSFAYWGQGTLVTVGAGATGTTAATACTGACTATAATGAGAAGTTC SA AAGGGCAAGGCCACGCTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGGTCAGCACCC TGACCTCTGAGGACTCTGCGATCTATTTCTGTGTCCTTTATTACTACGGTAGTTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCA208A-422H 51 GATGTACAGCTTCAGGAGTCAGGACCTGGCCT 52 208A-422HDVQLQESGPGLVNPSQSLSLTCS CGTGAATCCTTCTCAGTCTCTGTCTCTCACCTVTGYSITSGYYWIWIQQSPGNKL GCTCTGTCACTGGCTACTCCATCACCAGTGGTEWMGYIKYDGGNNYSPSLKNRIS TATTACTGGATCTGGATCCAGCAGTCTCCAGGIARDTSKNQCFLKLNSVTIEDTA AAACAAACTGGAATGGATGGGCTACATAAAGTTYYCTRGSDSFDYWGQGTTLTVS ACGACGGTGGCAATAACTACAGCCCATCTCTC SAAAAATCGAATCTCCATCGCTCGTGACACATC TAAGAACCAGTGTTTCCTGAAGTTGAATTCTGTGACTATTGAGGACACAGCTACATATTACTGT ACAAGAGGGTCGGACTCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 208A-442H 53GATGTACAGCTTCAGGAGTCAGGACCTGGCCT 54 208A-442H DVQLQESGPGLVNPSQSLSLTCSCGTGAATCCTTCTCAGTCTCTGTCTCTCACCT VTGYSITSGYYWIWIRQFPGNKLGCTCTGTCACTGGCTACTCCATCACCAGTGGT EWMGYIKYDGGNNYSPSLKNRISTATTACTGGATCTGGATCCGGCAGTTTCCAGG IARDTSKNQFFLKLNSVTIEDTAAAACAAACTGGAATGGATGGGCTACATAAAGT TYYCTRGSDSFDYWGQGTTLTVSACGACGGTGGCAATAACTACAGCCCATCTCTC S AAAAATCGAATCTCCATCGCTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTG TGACTATTGAGGACACAGCTACATATTACTGTACAAGAGGGTCGGACTCCTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA 208B-862H55 GATGTGCAGCTTCAGGAGTCGGGACCTGGCCT 56 208B-862H DVQLQESGPGLVKPSQSLSLTCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCT VTGYSITSDYAWNWIRQFPGNKLGCACTGTCACTGGCTACTCAATCACCAGTGAT EWMGYISYSGTTVYSPSLKSRISTATGCCTGGAACTGGATCCGGCAGTTTCCTGG ITRDTSKNQFFLQLNSVTIEDSAAAACAAACTGGAGTGGATGGGCTACATAAGCT TYYCGGNYWGQGTLVTVSAACAGTGGTACCACTGTCTACAGCCCATCTCTC AAAAGTCGAATCTCCATCACTCGGGACACATCCAAAAACCAGTTCTTCCTGCAATTGAATTCTG TGACTATTGAGGACTCAGCCACGTATTATTGTGGGGGTAATTACTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCA 208A-967H 57CAGGTGCAGCTGGAGGAGTCAGGACCTGGCCT 58 208A-967H QVQLEESGPGLVAPSQSLSITCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTT VSGFSLTSYGVHWVRQPPGKGLEGCACTGTCTCTGGATTTTCATTAACCAGCTAT WLGVIWAVGSINYNSALMSRLSIGGTGTACACTGGGTTCGCCAGCCTCCAGGAAA SKDNSKSQVFLKMNSLRTDDTAMGGGTCTGGAGTGGCTGGGAGTAATATGGGCTG YYCARDRTTATPFFDYWGQGTTLTTGGAAGTATAAATTATAATTCGGCTCTCATG TVSS TCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTAC GAACTGATGACACAGCCATGTACTACTGTGCCAGAGATCGGACTACGGCTACCCCCTTCTTTGA CTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCATCCAAAACA 208B-517H 59 CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCT 60208B-517H QVQLKQSGPGLVQPSQSLSITCT AGTGCAGCCCTCACAGAGCCTGTCCATCACCTVSGFSLITHGVHWVRQSPGKGLE GCACAGTCTCTGGTTTCTCATTAATTACCCATWLGVIWSGGSTDYNAAFISRLSI GGTGTACACTGGGTTCGCCAGTCTCCAGGAAASKDTSKSQVFLKMSSLQADDTAI GGGTCTGGAGTGGCTGGGAGTGATATGGAGTGYYCARNGGATAFDYWGQGTTLTV GTGGAAGCACAGACTATAATGCAGCTTTCATA SSTCCAGACTGAGCATCAGCAAGGACACCTCCAA GAGCCAAGTTTTCCTTAAAATGAGCAGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCC AGAAATGGGGGGGCTACGGCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B-822H 61CAGGTACAGCTGAAGCAGTCAGGACCTGGCCT 62 208B-822H QVQLKQSGPGLVQPSQSLSITCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCT VSGFSLITYGVHWVRQSPGKGLEGCACAGTCTCTGGTTTCTCATTAATTACCTAT WLGVIWGGGSTGYNAAFVSRLNIGGTGTACACTGGGTTCGCCAGTCTCCAGGAAA TKDNSKSQVFFKMNSLQPDDTAIGGGTCTGGAGTGGCTGGGAGTGATATGGGGTG YYCARNGGATVFDYWGQGTTLTVGTGGAAGCACAGGCTATAATGCAGCTTTCGTA SS TCCAGACTGAACATCACCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGC AACCTGATGACACAGCCATATACTACTGTGCCAGAAATGGAGGGGCTACGGTCTTTGACTACTG GGGCCAAGGCACCACTCTCACAGTCTCCTCA 208B-63 GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTT 64 208B-1024HEVQLVESGGDLVKPGGSLKLSCA 1024H AGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTASGFTFSNYGMSWVRQTPDHRLE GTGCAGCCTCTGGATTCACTTTCAGTAACTATWVATISSGGSYSYYPDSVKGRFT GGCATGTCTTGGGTTCGCCAGACTCCAGACAAISRDNAKNILYLQMSSLKSEDTA GAGGCTGGAGTGGGTCGCAACCATTAGTAGTGMYYCASLYYGYGDYWGQGTAFTV GTGGTAGTTATAGCTACTATCCAGACAGTGTA SSAAGGGGCGGTTCACCATCTCCAGAGACAATGC CAAGAACATCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGT GCAAGTCTCTACTACGGCTACGGGGACTACTGGGGCCAAGGCACCGCTTTCACAGTCTCCTCA 208B-327H 65GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTT 66 208B-327H EVQLVESGGGLVKPGGSLKLSCAGGTGAAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFTFSDSYMYWVRQTPDQRLEGTGCAGCCTCTGGATTCACTTTCAGTGACTCT WVATISDGGSYTFYPDSVKGRFTTATATGTATTGGGTTCGCCAGACTCCGGACCA ISRDNAQNNLYLQMSSLKSEDTAGAGGCTGGAGTGGGTCGCAACCATTAGTGATG MYYCASPHAGYFGWFAYWGRGTLGTGGTAGTTACACCTTCTATCCAGACAGTGTG VTVSA AAGGGACGATTCACCATCTCCAGAGACAATGCCCAGAACAACCTGTACCTGCAAATGAGCAGTC TGAAGTCTGAGGACACAGCCATGTATTACTGTGCATCCCCCCATGCTGGCTACTTCGGCTGGTT TGCTTACTGGGGCCGAGGGACTCTGGTCACTGTCTCTGCA 208B-353H 67 GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTT 68 208B-353HEVQLVESGGDLVKPGGSLKLSCA AGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTASGFTFNHYGMSWVRQPPDKRLE GTGCAGCCTCTGGATTCACTTTCAATCACTATWVATISSGGGYTYYPDSVKGRFT GGCATGTCTTGGGTTCGCCAGCCTCCAGACAAISRDNAKDTLSLQMSSLRSGDTA GAGACTGGAGTGGGTCGCAACCATTAGTAGTGVYYCASLYGSLFAYWGQGTLVTV GTGGTGGTTACACCTACTATCCAGACAGTGTG SAAAGGGGCGCTTCACCATCTCCAGAGACAATGC CAAGGACACCCTGTCCCTGCAAATGAGCAGTCTGAGGTCTGGGGACACAGCCGTGTATTACTGT GCAAGCCTATACGGTAGCCTGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 208B-178H 69GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTT 70 208B-178H DVKLVESGGGLVKLGGSLKLSCAAGTGAAGCTTGGAGGGTCCCTGAAACTCTCCT ASGFTFSSYYMSWVRQTPEKRLEGTGCAGCCTCTGGATTCACTTTCAGTAGCTAT LVAAINSNGGSTYYPDTVKGRFTTACATGTCTTGGGTTCGCCAGACTCCAGAGAA ISRDNAHNTLYLQMSSLKSEDTAGAGGCTGGAGTTGGTCGCAGCCATTAATAGTA LYYCARHGGLGRRDWYFDVWGAGATGGTGGTAGCACCTACTATCCAGACACTGTG TTVTVSSAAGGGCCGATTCACCATCTCCAGAGACAATGC CAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCTTGTATTACTGT GCAAGACATGGGGGACTGGGACGTAGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGG TCACCGTCTCCTCA 208B-672H 71GAAGTGAAACTGGTGGAGTCTGGGGGAAGTTT 72 208B-672H EVKLVESGGSLVQPGGSLKLSCAAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCT ASGFNFNTYAMSWVRQTPEKRLEGCGCAGCCTCTGGATTCAATTTCAATACCTAT WVAYISNGGGNTYYVDTVKGRFTGCCATGTCTTGGGTTCGCCAGACTCCAGAGAA ISRDNAHNTLYLRMSSLKSEDTAGAGGCTGGAGTGGGTCGCATACATTAGTAATG MYYCARHGLYWGYSMDYWGQGTSGTGGTGGTAACACCTACTATGTAGACACTGTA VTVSS AAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCGAATGAGCAGTC TGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATGGGCTCTACTGGGGCTATTCTAT GGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 208B-793H 73 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGTTT 74 208B-793HEVKLVESGGGLVQPGGSLKLSCA AGTGCAGCCAGGAGGGTCCCTGAAACTCTCCTASGFTFSSYAMSWVRQTPERRLE GTGCAGCCTCTGGATTCACTTTCAGTAGCTATWVTYISNGGGSTYYSDTVKGRFT GCCATGTCTTGGGTTCGCCAGACTCCAGAGAGFSRDNAHNTLYLQMSSLKSEDTA GAGGCTGGAGTGGGTCACATACATTAGTAATGMYYCARHGLGRTGFASWGQGTLV GTGGTGGTAGCACCTACTATTCAGACACTGTA TVSAAAGGGCCGATTCACCTTCTCCAGAGACAATGC CAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGT GCAAGACATGGACTGGGAAGGACAGGGTTTGCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCT CTGCA 208B-826H 75GATGTGCAGCTGGTGGAGTCTGGGGGAGGCCT 76 208B-826H DVQLVESGGGLVQAGGSRKLSCAAGTGCAGGCTGGAGGGTCCCGGAAACTCTCCT ASGFPFSSFGMHWVRQAPEKGLEGTGCAGCCTCTGGATTCCCTTTCAGTTCCTTT WVASISSRTSKIYYADNLKGRFTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAA ISRDNPRNTLFLQMTSLGSEDTAGGGGCTGGAGTGGGTCGCCTCCATTAGTAGTC MYYCVRSVFGNSYWFFDVWGAGTGCACTAGTAAGATCTACTATGCAGACAACCTG TVTVSS AAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTC TTGGATCTGAGGACACGGCCATGTATTACTGTGTAAGATCCGTCTTTGGTAATTCTTACTGGTT TTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA 208B-174H 77 GAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTT 78 208B-174HEVKLEESGGGLVQPGGSMKLSCV GGTACAACCTGGGGGATCCATGAAACTCTCCTASGFSFSSYWMSWVRQSPEKGLD GTGTAGCCTCTGGATTTTCTTTCAGTAGCTACWVAEIRLRSDNYATHYAESVKGR TGGATGTCTTGGGTCCGCCAGTCTCCAGAGAAFTISRDDSISRLYLQMNTLRAED GGGGCTTGACTGGGTTGCTGAAATTAGATTGATGIYYCTWMTYWGQGTLVTVSA GATCTGATAATTATGCAACCCATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGA TGATTCCATAAGTCGTCTCTACCTGCAAATGAACACCTTAAGAGCTGAAGACACTGGAATTTAT TACTGTACATGGATGACGTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAACA 208B-408H 79 GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTT80 208B-408H EVKLVESGGGLVQPGGSLRLSCA GGTACAGCCTGGGGGTTCTCTGAGACTCTCCTSSGFTFTDYYMSWVRQPPGKALE GTGCAAGTTCTGGGTTCACCTTCACTGATTACWLGFIRNKAYGYTTEFSASVNGR TACATGAGCTGGGTCCGCCAGCCTCCAGGAAAFTISRDDSQSVPYLQMNTLRAED GGCACTTGAGTGGTTGGGTTTTATTAGAAACASATYYCARVLYYDYGGFAYWGQG AAGCTTATGGTTACACGACCGAGTTCAGTGCA TLVTVSTTCTGTGAACGGTCGGTTCACCATCTCCAGAGA TGATTCCCAAAGCGTCCCCTATCTTCAAATGAACACCCTGAGAGCTGAGGACAGTGCCACTTAT TACTGTGCGAGAGTCCTCTACTATGATTACGGGGGATTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTACA 208B-612H 81GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCT 82 208B-612H EVQLQQSGPELVKPGASVMSCKGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCT ASGYRFTSYVMHWVRQKPGQGLEGTAAGGCTTCCGGATACAGATTCACTAGCTAT WIGYIDPHND--GTTATGCACTGGGTGAGGCAGAAGCCTGGACA DTKYSEKFRGKATLTSDKSSTTAGGGCCTTGAGTGGATTGGATATATTGATCCTC YMELSSLTSEDSAVYYCVRYSYDACAATGATGATACAAAATACAGTGAGAAGTTC RDYSPMDYWGQGTSVTVSSAGAGGTAAGGCCACACTGACTTCAGACAAGTC CTCCACCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGT GTGAGATATTCTTACGACAGGGATTACAGTCCTATGGACTACTGGGGTCAAGGAACCTCAGTCA CCGTCTCCTCA 208A-877H 83CAGCTGCAACAGTCTGGACCTGAGCTGGTGAA 84 208A-877H QLQQSGPELVKPGASVKISCKTSGCCTGGGGCTTCAGTGAAAATTTCCTGCAAGA GYTFTENAMHWVKQSRGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTVYTQKFKGKATLTCACTGGGTGAAGCAGAGCCGTGGAAAGAGCCT VAKSSSTAYMELRTMTCEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCASREPDFFDYWGQGSSVTVSSGTGATACTGTCTACACCCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGCCAAGTCTTCCAGCACAGCCTACATGGAGCTCCGCACCATGACAT GTGAGGAATCTACAGTGTATTACTGTGCAAGCCGGGAACCGGACTTCTTTGACTACTGGGGCCA AGGCTCCTCTGTCACAGTCTCCTCA 208B-251H 85CAGCTGCAACAGTCTGGACCTGTACTGGTGAA 86 208B-251H QLQQSGPVLVKPGASVKISCKTSGCCTGGGGCTTCAGTGAAAATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIYNQKFKGKATLTCACTGGGTGAAGCAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESPVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG FCVRRELDFFDYWGQGTSVTVSSGTGATACTATCTACAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCCCAGTCTATTTCTGTGTAAGACGGGAACTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-110H 87CAGCTGCAACAGTCTGGCCCTGTCCTGGTGAA 88 208A-110H QLQQSGPVLVKSGTSVKISCKTS208B- GTCTGGGACTTCAGTTAAAATTTCCTGCAAGA 208B-1070HGYTFTENAMHWVKQSHGQSLEWI 1070H CTTCTGGATACACATTCACTGAAAACGCCATGGGVNPNNGDTIFNQKFKGKATLT CACTGGGTGAAACAGAGCCATGGACAGAGCCTVDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATGYCVRRELDFFDYWGQGTSVTVSS GTGATACTATCTTCAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAACTGGACTTCTTTGACTACTGGGGTCAAGGCACCTCTGTCACAGTCTCCTCA 208A-334H 89 CAGCTGCAACAGTCTGGCCCTGTCCTGGTGAA90 208A-334H QLQQSGPVLVKPGTSVKISCKTS 208A-920HGCCTGGGACTTCAGTGAAAATTTCCTGCAAGA 208A-920H GYTFTENAMHWVKQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLTCACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSSGTGATACTATCTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGACGGGAACTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-159H 91CAGCTGCAACAGTCTGGACCTGCCCAGGTGAA 92 208A-159H QLQQSGPAQVKPGASVMISCKTSGCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKDKAALTCACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMELRSLTSEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSSGTGATACTATTTTCAACCAGAAGTTCAAGGAC AAGGCCGCATTGACTGTAGACAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACAT CTGAGGAGTCCACAGTCTATTACTGTGTAAGACGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-126H 93CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 94 208A-126H QLQQSGPVLVKPGASVMISCKTSGCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLTCACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSNTAFTELRSLPSEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRGLDFFDYWGQGTSVTVSSGTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAACACAGCCTTCACGGAGCTCCGCAGCCTGCCAT CTGAAGAATCCACAGTCTATTACTGTGTTAGACGGGGATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208A-133H 95CAGCTGCAACAGTCTGGCCCTGTCCTGGTGGG 96 208A-133H QLQQSGPVLVGPGASVMISCKTSGCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVEQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLTCACTGGGTGGAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMEFRSLTSEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSSGTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTTCATGGAGTTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGACGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCA 208B-556H 97CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 98 208B-556H QLQQSGPVLVKPGASVMISCKTSGCCTGGGGCTTCAGTGATGATTTCCTGCAAGA GYTFTENAMHWVKQSHGKSLEWICTTCTGGATACACATTCACTGAAAACGCCATG GGVNPNNGDTIFNQHFKGKATLTCACTGGGTGAAACAGAGCCATGGAAAGAGCCT VDKSSSTAFMEFRSLTSEESTVYTGAGTGGATTGGAGGTGTTAATCCTAACAATG YCVRRELDFFDYWGQGTSVTVSSGTGATACTATTTTCAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTTCATGGAGTTCCGCAGCCTGACAT CTGAAGAATCCACAGTCTATTACTGTGTAAGACGGGAATTGGACTTCTTTGACTACTGGGGCCA AGGCACCTCTGTCACAGTCTCCTCAACA 208A-741H99 CAGCTGCAACAGTCTGGACCTGTCCTGGTGAA 100 208A-741HQLQQSGPVLVKPGASVMISCKTS GCCTGGGGCTTCAGTGATGATTTCCTGCAAGAGYTFTENAMHWVKQSHGKSLEWI CTTCTGGATACACATTCACTGAAAACGCCATGGGVNPNNGDTIFNQHFKGKATLT CACTGGGTGAAACAGAGCCATGGAAAGAGCCTVDKSSSTAFMELRSLTSEESTVY TGAGTGGATTGGAGGTGTTAATCCTAACAATGYCVRRELDFFDYWGQGTSVTVSS GTGATACTATTTTCAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAAGAATCCACAGTCTATTACTGTGTAAGA CGGGAATTGGACTTCTTTGACTACTGGGGCCAAGGCACCTCTGTCACAGTCTCCTCA 208A-1064 101 GAATACGCCATGCAC 102 208A-1064EYAMH CDR H1 CDR H1 208B-1094 208B-1094 CDR H1 CDR H1 208A-293 208A-293CDR H1 CDR H1 208A-222 208A-222 CDR H1 CDR H1 208B-560 208B-560 CDR H1CDR H1 208A-605 103 GAATACGCCATACAC 104 208A-605 EYAIH CDR H1 CDR H1208A-830 208A-830 CDR H1 CDR H1 208A-134 105 AGTAGTTATATAAGT 106208A-134 SSYIS CDR H1 CDR H1 208A-692 208A-692 CDR H1 CDR H1 208A-557208A-557 CDR H1 CDR H1 208B-281 208B-281 CDR H1 CDR H1 208A-352 107AGTAGTTTTATAAGT 108 208A-352 SSFIS CDR H1 CDR H1 208A-983 109AGTAGTTATTTTAGT 110 208A-983 SSYFS CDR H1 CDR H1 208B-471 111AGCAGTACCTATATAAGT 112 208B-471 STYIS CDR H1 CDR H1 208A-207 113AGCTACTGGATACAC 114 208A-207 SYWIH CDR H1 CDR H1 208A-638 208A-638CDR H1 CDR H1 208B-515 208B-515 CDR H1 CDR H1 208A-874 115AGCTATTGGATGCAC 116 208A-874 SYWMH CDR H1 CDR H1 208B-911 117CCTACTCGATAAAC 118 208B-911 TYSIN CDR H1 CDR H1 208B-1096 119GCCAACAAGATGCAC 120 208B-1096 ANKMH CDR H1 CDR H1 208B-589 208B-589CDR H1 CDR H1 208B-395 208B-395 CDR H1 CDR H1 208B-189 208B-189 CDR H1CDR H1 208B-547 121 AGCAACAAGATGCAC 122 208B-547 SNKMH CDR H1 CDR H1208A-210 123 AGCTACTATGTACAC 124 208A-210 SYYVH CDR H1 CDR H1 208A-422125 AGTGGTTATTACTGGATC 126 208A-422 SGYYWI CDR H1 CDR H1 208A-442208A-442 CDR H1 CDR H1 208B-862 127 AGTGATTATGCCTGGAAC 128 208B-862SDYAWN CDR H1 CDR H1 208A-967 129 AGCTATGGTGTACAC 130 208A-967 SYGVHCDR H1 CDR H1 208B-517 131 ACCCATGGTGTACAC 132 208B-517 THGVH CDR H1CDR H1 208B-822 133 ACCTATGGTGTACAC 134 208B-822 TYGVH CDR H1 CDR H1208B-1024 135 AACTATGGCATGTCT 136 208B-1024 NYGMS CDR H1 CDR H1 208B-327137 GACTCTTATATGTAT 138 208B-327 DSYMY CDR H1 CDR H1 208B-353 139CACTATGGCATGTCT 140 208B-353 HYGMS CDR H1 CDR H1 208B-178 141AGCTATTACATGTCT 142 208B-178 SYYMS CDR H1 CDR H1 208B-672 143ACCTATGCCATGTCT 144 208B-672 TYAMS CDR H1 CDR H1 208B-793 145AGCTATGCCATGTCT 146 208B-793 SYAMS CDR H1 CDR H1 208B-826 147TCCTTTGGAATGCAC 148 208B-826 SFGMH CDR H1 CDR H1 208B-174 149AGCTACTGGATGTCT 150 208B-174 SYWMS CDR H1 CDR H1 208B-408 151GATTACTACATGAGC 152 208B-408 DYYMS CDR H1 CDR H1 208B-612 153AGCTATGTTATGCAC 154 208B-612 SYVMH CDR H1 CDR H1 208A-877 155GAAAACGCCATGCAC 156 208A-877 ENAMH CDR H1 CDR H1 208B-251 208B-251CDR H1 CDR H1 208A-110 208A-110 CDR H1 CDR H1 208B-1070 208B-1070 CDR H1CDR H1 208A-334 208A-334 CDR H1 CDR H1 208A-920 208A-920 CDR H1 CDR H1208A-159 208A-159 CDR H1 CDR H1 208A-126 208A-126 CDR H1 CDR H1 208A-133208A-133 CDR H1 CDR H1 208B-556 208B-556 CDR H1 CDR H1 208A-741 208A-741CDR H1 CDR H1 208A-1064 157 GGTATCAATCCTACTAATGGTGATACAATCTA 158208A-1064 GINPTNGDTIYNQHFKD CDR H2 CAACCAGAAGTTCAAGGAC CDR H2 208A-293208A-293 CDR H2 CDR H2 208B-1094 159 GGTATCAATCCTACTAATGGTGATACAATCTA160 208B-1094 GINPTNGDTIYNQRFKD CDR H2 CAACCAGAGGTTCAAGGAC CDR H2208A-222 161 GGTATTAATCCTAACAATGGCAATGCTATCTA 162 208A-222GINPNNGNAIYNQIFKD CDR H2 CAACCAGATATTCAAGGAC CDR H2 208A-605 163GGTATTAATCCTAGCAATGGCAATGCTATCTA 164 208A-605 GINPSNGNAIYNQIFKD CDR H2CAACCAAATATTCAAGGAC CDR H2 208B-560 208B-560 CDR H2 CDR H2 208A-830 165GGTATTAATCCTAGCAATGGCGATCCTATCTA 166 208A-830 GINPSNGDPITNQIFKD CDR H2TAACCAGATATTCAAGGAC CDR H2 208A-134 167 TGGATTTATGCTGGAACTGGTAATACTAACTA168 208A-134 WIYAGTGNTNYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2208A-557 208A-557 CDR H2 CDR H2 208A-352 208A-352 CDR H2 CDR H2 208A-692169 TGGATTTTTGCTGGAACTGGTAATACTAATTA 170 208A-692 WIFAGTGNTNYNQKFTDCDR H2 TAATCAGAAGTTCACAGAC CDR H2 208A-983 171TGGATTTATGCTGGAACTGGTAATACTATCTA 172 208A-983 WIYAGTGNTIYNQKFTD CDR H2TAATCAGAAGTTCACAGAC CDR H2 208B-281 173 TGGATTTATGCTGGAACTGGTGGTACTAACTA174 208B-281 WIYAGTGGTNYNQKFTD CDR H2 TAATCAGAAGTTCACAGAC CDR H2208B-471 175 TGGATTTATGCTGGAACTGGTGCTACTAATTA 176 208B-471WIYAGTGATNYNQKFTG CDR H2 TAATCAGAAGTTCACAGGC CDR H2 208A-207 177GAAATTGATCCTTCTGACAGTTATATTAACCA 178 208A-207 EIDPSDSYINQNQHFRG CDR H2GAATCAAAAGTTCAGGGGC CDR H2 208A-638 179 GAAGTTGATCCTTCTGACAGTTATATTAACCA180 208A-638 EVDPSDSYINQNEKERG CDR H2 GAATGAAAAGTTCAGGGGC CDR H2208B-515 181 GAGATTGATCCTTCTGATAGTTATACTAACTA 182 208B-515EIDPSDSYTNYNQHFKG CDR H2 CAATCAAAAGTTCAAGGGC CDR H2 208A-874 183GAGATTCATCCTTCTGATAGTTATACTAGCTA 184 208A-874 EIHPSDSYTSYNQHFKD CDR H2CAATCAAAAGTTCAAGGAC CDR H2 208B-911 185 AATATTTATCCTTCTACCAGTCATACTAACTA186 208B-911 NIYPSTSHTNYNQKFRD CDR H2 CAATCAAAAGTTCAGGGAC CDR H2208B-1096 187 TACATTGATCCTAGCTCTGGTTATACTGAATA 188 208B-1096YIDPSSGYTEYNHKIQY CDR H2 CAATCATAAGATCCAGTAC CDR H2 208B-589 189TACATTGATCCTAGCTCTGGTTATACTGAATA 190 208B-589 YIDPSSGYTEYNHKIQD CDR H2CAATCATAAGATCCAGGAC CDR H2 208B-395 191 TACATTGATCCTAGCTCTGGTTATACTCAATA192 208B-395 YIDPSSGYTQYNHKIQD CDR H2 CAATCATAAGATCCAGGAC CDR H2208B-189 193 TACATTGATCCTGCCTCTGGCTATACTGAATA 194 208B-189YIDPASGYTEYNQKIKD CDR H2 CAATCAGAAGATCAAGGAC CDR H2 208B-547 195TACATTGATCCTAGCTCTGGTTATACTGAATA 196 208B-547 YIDPSSGYTEYNQKIKD CDR H2CAATCAGAAGATCAAGGAC CDR H2 208A-210 197 TGTATTTATCCTGGAGATGTTAATACTGACTA198 208A-210 CIYPGDVNTDYNEKFKG CDR H2 TAATGAGAAGTTCAAGGGC CDR H2208A-422 199 TACATAAAGTACGACGGTGGCAATAACTACAG 200 208A-422YIKYDGGNNYSPSLKN CDR H2 CCCATCTCTCAAAAAT CDR H2 208A-442 208A-442 CDR H2CDR H2 208B-862 201 TACATAAGCTACAGTGGTACCACTGTCTACAG 202 208B-862YISYSGTTVYSPSLKS CDR H2 CCCATCTCTCAAAAGT CDR H2 208A-967 203GTAATATGGGCTGTTGGAAGTATAAATTATAA 204 208A-967 VIWAVGSINYNSALMS CDR H2TTCGGCTCTCATGTCC CDR H2 208B-517 205 GTGATATGGAGTGGTGGAAGCACAGACTATAA206 208B-517 VIWSGGSTDYNAAFIS CDR H2 TGCAGCTTTCATATCC CDR H2 208B-822207 GTGATATGGGGTGGTGGAAGCACAGGCTATAA 208 208B-822 VIWGGGSTGYNAAFVSCDR H2 TGCAGCTTTCGTATCC CDR H2 208B-1024 209ACCATTAGTAGTGGTGGTAGTTATAGCTACTA 210 208B-1024 TISSGGSYSYYPDSVKG CDR H2TCCAGACAGTGTAAAGGGG CDR H2 208B-327 211 ACCATTAGTGATGGTGGTAGTTACACCTTCTA212 208B-327 TISDGGSYTFYPDSVKG CDR H2 TCCAGACAGTGTGAAGGGA CDR H2208B-353 213 ACCATTAGTAGTGGTGGTGGTTACACCTACTA 214 208B-353TISSGGGYTYYPDSVKG CDR H2 TCCAGACAGTGTGAAGGGG CDR H2 208B-178 215GCCATTAATAGTAATGGTGGTAGCACCTACTA 216 208B-178 AINSNGGSTYYPDTVKG CDR H2TCCAGACACTGTGAAGGGC CDR H2 208B-672 217 TACATTAGTAATGGTGGTGGTAACACCTACTA218 208B-672 YISNGGGNTYYVDTVKG CDR H2 TGTAGACACTGTAAAGGGC CDR H2208B-793 219 TACATTAGTAATGGTGGTGGTAGCACCTACTA 220 208B-793YISNGGGSTYYSDTVKG CDR H2 TTCAGACACTGTAAAGGGC CDR H2 208B-826 221TCCATTAGTAGTCGCACTAGTAAGATCTACTA 222 208B-826 SISSRTSKITYADNLKG CDR H2TGCAGACAACCTGAAGGGC CDR H2 208B-174 223 GAAATTAGATTGAGATCTGATAATTATGCAAC224 208B-174 EIRLRSDNYATHYAESVKG CDR H2 CCATTATGCGGAGTCTGTGAAAGGG CDR H2208B-408 225 TTTATTAGAAACAAAGCTTATGGTTACACGAC 226 208B-408FIRNKAYGYTTEFSASVNG CDR H2 CGAGTTCAGTGCATCTGTGAACGGT CDR H2 208B-612 227TATATTGATCCTCACAATGATGATACAAAATA 228 208B-612 YIDPHNDDTKYSEKFRG CDR H2CAGTGAGAAGTTCAGAGGT CDR H2 208A-877 229 GGTGTTAATCCTAACAATGGTGATACTGTCTA230 208A-877 GVNPNNGDTVYTQKFKG CDR H2 CACCCAGAAGTTCAAGGGC CDR H2208B-251 231 GGTGTTAATCCTAACAATGGTGATACTATCTA 232 208B-251GVNPNNGDTIYNQKFKG CDR H2 CAACCAGAAGTTCAAGGGC CDR H2 208A-110 233GGTGTTAATCCTAACAATGGTGATACTATCTT 234 208A-110 GVNPNNGDTIFNQKFKG CDR H2CAACCAGAAGTTCAAGGGC CDR H2 208B-1070 208B-1070 CDR H2 CDR H2 208A-334208A-334 CDR H2 CDR H2 208A-920 208A-920 CDR H2 CDR H2 208A-126 208A-126CDR H2 CDR H2 208A-133 208A-133 CDR H2 CDR H2 208B-556 208B-556 CDR H2CDR H2 208A-741 208A-741 CDR H2 CDR H2 208A-159 235GGTGTTAATCCTAACAATGGTGATACTATTTT 236 208A-159 GVNPNNGDTIFNQKFKD CDR H2CAACCAGAAGTTCAAGGAC CDR H2 208A-1064 237 CGGGAACTGGACTACTTTGCCTCC 238208A-1064 RELDYFAS CDR H3 CDR H3 208B-1094 208B-1094 CDR H3 CDR H3208A-293 239 CGGGAACTGGACTACTTTCCCTCC 240 208A-293 RELDYFPS CDR H3CDR H3 208A-222 241 CGACAACTGGACTACTTTGACTAT 242 208A-222 RQLDYFDYCDR H3 CDR H3 208A-605 243 CGACAACTGGACTTCTTTGACTAT 244 208A-605RQLDFFDY CDR H3 CDR H3 208B-560 208B-560 CDR H3 CDR H3 208A-830 245CGACAACTGGACTACTTTGACTTT 246 208A-830 RQLDYFDF CDR H3 CDR H3 208A-134247 AGTGGGACGGGGTTTACTTAC 248 208A-134 SGTGFTY CDR H3 CDR H3 208A-692208A-692 CDR H3 CDR H3 208A-557 208A-557 CDR H3 CDR H3 208A-352 208A-352CDR H3 CDR H3 208A-983 208A-983 CDR H3 CDR H3 208B-281 249AGTGGGACGGGGTTTATTTAC 250 208B-281 SGTGFIY CDR H3 CDR H3 208B-471 251TCTGGGGCGGGGGTTTAC 252 208B-471 SGAGVY CDR H3 CDR H3 208A-207 253CATTACTACGGTGTTCTTGACTCC 254 208A-207 HYYGVLDS CDR H3 CDR H3 208A-638208A-638 CDR H3 CDR H3 208B-515 255 CATTACTACGGTGTCTTTGACTCC 256208B-515 HYYGVFDS CDR H3 CDR H3 208A-874 257GGGGGCTACTATAGGTACGACGAGTTTGCTTA 258 208A-874 GGYYRYDEFAY CDR H3 CCDR H3 208B-911 259 AATGCCTATTCTATGGACTAC 260 208B-911 NAYSMDY CDR H3CDR H3 208B-1096 261 TTTGCTTAC 262 208B-1096 FAY CDR H3 CDR H3 208B-589208B-589 CDR H3 CDR H3 208B-395 208B-395 CDR H3 CDR H3 208B-189 208B-189CDR H3 CDR H3 208B-547 208B-547 CDR H3 CDR H3 208A-210 263TATTACTACGGTAGTTTTGCTTAC 264 208A-210 YYYGSFAY CDR H3 CDR H3 208A-422265 GGGTCGGACTCCTTTGACTAC 266 208A-422 GSDSFDY CDR H3 CDR H3 208A-442208A-442 CDR H3 CDR H3 208B-862 267 AATTAC 268 208B-862 NY CDR H3 CDR H3208A-967 269 GATCGGACTACGGCTACCCCCTTCTTTGACTA 270 208A-967 DRTTATPFFDYCDR H3 C CDR H3 208B-517 271 AATGGGGGGGCTACGGCCTTTGACTAC 272 208B-517NGGATAFDY CDR H3 CDR H3 208B-822 273 AATGGAGGGGCTACGGTCTTTGACTAC 274208B-822 NGGATVFDY CDR H CDR H 208B-1024 275 CTCTACTACGGCTACGGGGACTAC276 208B-1024 LYYGYGDY CDR H3 CDR H3 208B-327 277CCCCATGCTGGCTACTTCGGCTGGTTTGCTTA 278 208B-327 PHAGYFGWFAY CDR H3 CCDR H3 208B-353 279 CTATACGGTAGCCTGTTTGCTTAC 280 208B-353 LYGSLFAYCDR H3 CDR H3 208B-178 281 CATGGGGGACTGGGACGTAGGGACTGGTACTT 282 208B-178HGGLGRRDWYFDV CDR H3 CGATGTC CDR H3 208B-672 283CATGGGCTCTACTGGGGCTATTCTATGGACTA 284 208B-672 HGLYWGYSMDY CDR H3 CCDR H3 208B-793 285 CATGGACTGGGAAGGACAGGGTTTGCTTCC 286 208B-793HGLGRTGFAS CDR H3 CDR H3 208B-826 287 TCCGTCTTTGGTAATTCTTAC 288 208B-826SVFGNSYWFFDV CDR H3 CDR H3 208B-174 289 ATGACGTAC 290 208B-174 MTYCDR H3 CDR H3 208B-408 291 GTCCTCTACTATGATTACGGGGGATTTGCTTA 292 208B-408VLYYDYGGFAY CDR H3 C CDR H3 208B-612 293TATTCTTACGACAGGGATTACAGTCCTATGGA 294 208B-612 YSYDRDYSPMDY CDR H3 CTACCDR H3 208A-877 295 CGGGAACCGGACTTCTTTGACTAC 296 208A-877 REPDFFDYCDR H3 CDR H3 208B-251 297 CGGGAATTGGACTTCTTTGACTAC 298 208B-251RELDFFDY CDR H3 CDR H3 208A-110 208A-110 CDR H3 CDR H3 208B-1070208B-1070 CDR H3 CDR H3 208A-334 208A-334 CDR H3 CDR H3 208A-920208A-920 CDR H3 CDR H3 208A-159 208A-159 CDR H3 CDR H3 208A-133 208A-133CDR H3 CDR H3 208B-556 208B-556 CDR H3 CDR H3 208A-741 208A-741 CDR H3CDR H3 208A-126 299 CGGGGATTGGACTTCTTTGACTAC 300 208A-126 RGLDFFDYCDR H3 CDR H3 208A- 301 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 302 208A-1064LDIVLTQSPASLIVSLGQTATISC 1064L AATTGTTTCTCTGGGGCAGACGGCCACCATCTRASQSVSTSRFSYLHWIQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCTPPKLLIKYASNLESGVPVRFSGS AGGTTTAGTTATCTGCACTGGATCCAACAGAAGSGTDFTLNIHPVEEEDTATYFC ACCAGGGCAGCCACCCAAACTCCTCATCAAGTQHSWEFPFTFGSGTKLKIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGTCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTTCTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGAAAATAAAACGGGCTGATGCTGC 208B- 303 GACATTGTGCTGACACAGTCTCCTGCTTCCTT304 208B-1094L DIVLTQSPASLIVSLGQTATISC 1094LAATTGTTTCTCTGGGGCAGACGGCCACCATCT RASQSLSTSRFSYVHWIQQFPGQCATGCAGGGCCAGCCAAAGTCTCAGTACATCT PPKLLIKYASNLESGVPVRFSGSAGGTTTAGCTATGTGCACTGGATCCAACAGAA GSGTDFTLNIHPVEEEDTATYFCACCAGGGCAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLKIKRATGCATCCAACCTTGAATCTGGGGTCCCTGTC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAACATATTTCTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGAAAATAAAACGGGCTGATGCTGC 208A-293L305 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 306 208A-293LDIVLTQSPASLIVSLGQRATISC AATTGTATCTCTGGGGCAGAGGGCCACCATCTRASQSVSTSRFSYVHWIQQFPGQ CATGTAGGGCCAGCCAAAGTGTCAGTACATCCPPKLLIKYASNLESGVPVRFSGS AGGTTTAGTTATGTGCACTGGATCCAACAGAAGSGTDFILNIHPVEEEDTATYFC ACCAGGGCAGCCACCCAAACTCCTCATCAAGTQHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTTGAATCTGGGGTCCCTGTCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CATCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTTCTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGC 208A-134L 307GAAGTTTTGATGACCCAAAGTCCACTCTCCCT 308 208A-134L EVLMTQSPLSLPVSLGDQASISCGCCTGTCAGTCTTGGAGATCAGGCCTCCATCT RSSQSLEHTNGNTYLEWFLQRPGCTTGCAGATCTAGTCAGAGCCTTGAACATACT QPPKLLITKVSSRFSGVPDRFSGAATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYYGAGACCAGGCCAGCCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLAIKRACAAAGTTTCCAGCCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGCAATAAAACGGGCTGATGCTGC208A-692L 309 GAAGTTTTGATGACCCAAAGTCCACTCTCCCT 310 208A-692LEVLMTQSPLSLPVSLGDQASISC GCCTGTCAGTCTTGGAGATCAGGCCTCCATCTRSSQSLEHSNGNTYLEWFLQRPG CTTGCAGATCTAGTCAGAGCCTTGAACATAGTQPPKLLIYKVSSRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCASGSGTDFTLKISRVEAEDLGVYY GAGACCAGGCCAGCCTCCAAAGCTCCTGATCTCFQGSHVPFTFGSGTKLAIKR ACAAAGTTTCCAGCCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGCAATAAAACGGGCTGATGCTGC 208A-352L 311GATGTTTTGATGGCCCAAACTCCACTCTCCCT 312 208A-352L DVLMAQTPLSLPVTLGDQASISCGCCTGTCACCCTTGGAGATCAAGCCTCCATCT RSSQSLVHSNGNTYLEWFLQKPGCTTGCAGATCTAGTCAGAGCCTTGTACATAGT QSPKLLIYNVSNRFSGVPDRFSGAATGGAAACACCTATTTAGAGTGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYYGAAACCAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPLTFGSGTKLEIKRACAACGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCACTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAACGGGCTGATGCTGC208A-983L 313 GATGTTTTGATGACCCAAACTCCACTCTCCCT 314 208A-983LDVLMTQTPLSLPVNLGDQASISC GCCTGTCAATCTTGGAGATCAGGCCTCCATCTRSSQSLLHSNGNTYLEWFLQKPG CTTGCAGATCTAGTCAGAGCCTTCTACATAGTQSPKLLIYNVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTTCCTGCASGSGTDFTLKISRVEAEDLGVYY GAAACCAGGCCAGTCTCCAAAGCTCCTGATCTCFQGSHVPFTFGSGTKLAIKR ACAATGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGCAATAAAACGGGCTGATGCTGC 208B-281L 315GATGTTTTGATGACCCAAACTCCACTCTCCCT 316 208B-281L DVLMTQTPLSLPVSLGDQASISCGCCTGTCAGTCTTGGAGATCAAGCCTCCATCT RSSQSLVHNNGNTYLEWFLQKPGCTTGCAGATCTAGTCAGAGCCTTGTACATAAT QSPKLLIYKVSNRFSGVPDRFSGAATGGAAACACCTATTTAGAATGGTTCCTGCA SGSGTDFTLKISRVEAEDLGVYYGAAACCAGGCCAGTCTCCAAAGCTCCTGATCT CFQGSHVPFTFGSGTKLEIKRACAAAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAACGGGCTGATGCTGC 208B-317 GATGTTTTGATGACCCAAACTCCACTCTCCCT 318 208B-1024LDVLMTQTPLSLPVSLGDQASISC 1024L GCCTGTCAGTCTTGGAGATCAAGCCTCCATCTRSSQSIVHSNGNTYLEWYLQKLG CTTGCAGATCTAGTCAGAGCATTGTACACAGTQSPKLLITRVSNRFSGVPDRFSG AATGGAAACACCTATTTAGAGTGGTACCTGCASGSGTDFTLKISRVEAEDLGVYY GAAACTAGGCCAGTCTCCAAAGCTCCTGATCTCFQGSHVPWTFGGGTKLEIKR ACAGAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGT TCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGC 208B-471L 319GATGTTGTGATGACCCAAACTCCACTCTCCCT 320 208B-471L DVVMTQTPLSLPVSLGDQASISCGCCTGTCAGTCTTGGAGATCAAGCCTCCATCT RSSQSLLHSNGNTYLEWYLQKPGCTTGCAGATCTAGTCAGAGCCTTCTACATAGT QSPNLLIYNVSNRFSGVPDRFSGAATGGAAACACCTATTTAGAATGGTACCTGCA SGSGTDFALKISRVGAEDLGVYYGAAACCTGGCCAGTCTCCAAACCTCCTGATCT CFQGSHVPLTFGAGTKLELKRACAATGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCGCACTCAAGATCAGCAGAGTGGGGGCTG AGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGAC CAAGCTGGAGCTGAAACGGGCTGATGCTGC208B-862L 321 GATGTTGTGCTGACCCAAACTCCACTCTCCCT 322 208B-862LDVVLTQTPLSLPFSFGNKASIFC GCCTCTCAGTCTTGGAGATCAGGCCTCCATCTKFSQTLLHRDGNPFLLWYLQKPG CTTGCAGATCTAGTCAGACCCTTCTACACAGTQSPKLLIYKLSNRFFGVPKRFRG GATGGAGACACCTATTTACATTGGTACCTGCARGSGTNFPLKISKGEAEDLGVFF GAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTCSQSTHVPYTFGGGTKLEIKR ACAAACTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGT ACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGC 208B-589L 323GATATTGTGATGACCCAGACTCCACTCACTTT 324 208B-589L DIVMTQTPLTLSVTIGQPASISC208B- GTCGGTTACCATTGGACAACCAGCTTCCATCT 208B-1096LKSSQSLLFTNGKTYLNWFLQRPG 1096L CTTGCAAGTCAAGTCAGAGCCTCTTATTTACTQSPKRLIYLLSKLDSGVPDRFSG AATGGAAAAACCTATTTAAATTGGTTTTTACASGSGTDFTLKISRVEAEDLGVYY GAGGCCAGGCCAGTCTCCAAAACGCCTAATCTCLQSTYFPLTFGAGTKLELKR ATCTGCTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGGACAGA TTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCTTGCAGAGT ACATATTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGC 208B-189L 325GATGTTGTGATGACCCAGACTCCACTCACTTT 326 208B-189L DVVMTQTPLTLSVTIGQPASISCGTCGGTTACCATTGGACAACCAGCTTCCATCT KSSQSLLYTNGKTYLNWLLQRPGCTTGCAAGTCAAGTCAGAGCCTCTTATATACT QSPKRLIYLVSKLDSGVPDRFSGAATGGAAAGACCTATTTGAATTGGTTATTACA SGSGTDFTLKISRVEAEDLGVYYGAGGCCAGGCCAGTCTCCAAAACGCCTAATCT CLQSIHFPYTFGGGTKLDIKRATCTGGTGTCAAAATTGGACTCTGGAGTCCCT GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTG AGGATTTGGGAGTTTATTACTGCTTGCAGAGTATACATTTTCCGTACACGTTCGGAGGGGGGAC CAAGCTGGACATAAAACGGGCTGATGCTGC208B-327L 327 GATATTGTGATGACGCAGGCTGCATTCTCCAA 328 208B-327LDIVMTQAAFSNPVTPGTSVSISC TCCAGTCACTCCTGGAACATCAGTTTCCATCTRSSKSLLHSNGITYLYWYLQKPG CCTGCAGGTCTAGTAAGAGTCTCCTACATAGTQSPQLLIYQMSKIASGVPDRFRS AATGGCATCACTTATTTGTATTGGTATCTGCASGSGTDFTLRISRVEAADVGVYY GAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTCAQNLELPWTFGGGTKLEIKR ATCAGATGTCCAAGATTGCCTCAGGAGTCCCAGACAGGTTCAGGAGCAGTGGGTCAGGAACTGA TTTCACACTGAGAATCAGCAGAGTGGAGGCTGCGGATGTGGGTGTTTATTACTGTGCTCAAAAT CTAGAACTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGC 208A-874L 329GACATTGTGATGACCCAGTCTCACAAATTCAT 330 208A-874L DIVMTQSHKFMSTSIGDRVSITCGTCCACATCAATAGGAGACAGGGTCAGCATCA KASQNVSPAVAWYQQKPGQSPKLCCTGCAAGGCCAGTCAGAATGTGAGTCCTGCT LIYSASSRYTGVPDRFTGSGSGTGTAGCCTGGTATCAACAGAAACCAGGACAATC AFTFTISSVQAEDLAVYFCQQHFTCCTAAACTACTGATTTACTCGGCATCCTCCC STPWTFGGGTMLEIKRGATACACTGGAGTCCCTGATCGCTTCACTGGC AGTGGATCTGGGACGGCTTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTTCTGTCAGCAACATTTTAGTACTCCGTGGACGTTCGGTGGAGGCACCATGCTGGAAATCAA ACGGGCTGATGCTGC 208B-353L 331GACATTGTGATGACCCAGTCTCAAAAATTCAT 332 208B-353L DIVMTQSQKFMSTTVGDRVRVTCGTCCACAACAGTTGGGGACAGGGTCAGAGTCA KASQNVGTAVAWYQQKPGQSPKLCCTGCAAGGCCAGTCAGAATGTGGGTACTGCT LIYSASNRYTGVPDRFTGSGSGTGTAGCCTGGTATCAACAGAAACCAGGACAATC DFTLTITNMQSEDLADYFCQQYSTCCTAAACTACTGATTTACTCAGCATCCAATC TYPLTFGSGAKLEIKRGGTACACTGGAGTCCCTGATCGCTTCACAGGC AGTGGATCTGGGACAGATTTCACTCTCACCATTACCAATATGCAGTCTGAAGACCTGGCAGATT ATTTCTGTCAGCAATATAGCACCTATCCTCTCACGTTCGGCTCGGGGGCAAAGTTGGAAATAAA ACGGGCTGATGCTGC 208B-793L 333GACATTGTGATGACCCAGTCTCACAAATTCAT 334 208B-793L DIVMTQSHKFMSTSVGDRVSITCGTCCACATCAGTAGGAGACAGGGTCAGCATCA KASQDVGTAVAWYQQKPGQSPKLCCTGCAAGGCCAGTCAGGATGTGGGTACTGCT LIYWASTRHTGVPDRFTGSGSGTGTAGCCTGGTATCAACAGAAACCAGGACAATC DFTLTISNVQSEDLADYFCQQYSTCCTAAACTACTGATTTACTGGGCATCCACCC NYLTFGAGTKLEVKRGGCACACTGGAGTCCCTGATCGCTTCACAGGC AGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATT ATTTCTGTCAGCAATATAGCAACTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGGTGAAACG GGCTGATGCTGC 208B-672L 335GACATCCAGATGACCCAGTCTCCATCCTCCTT 336 208B-672L DIQMTQSPSSLSASLGERVSLTCATCTGCCTCTCTGGGCGAAAGAGTCAGTCTCA RASQDIGGSINWLQQEPDGTIERCTTGTCGGGCAAGTCAGGACATTGGTGGTAGC LIYATSSLDSGVPKRFSGSRSGSATAAACTGGCTTCAGCAGGAACCAGATGGAAC DYSLTISSLESEDFVDYYCLQYATATTAAACGCCTGATCTACGCCACATCCAGTT SSPPTFGGGTKLEIKRTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGC AGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACT ATTACTGTCTACAATATGCTAGTTCTCCTCCGACGTTCGGTGGAGGCACCAAACTGGAAATCAA ACGGGCTGATGCTGC 208B-408L 337GACATCCAGATGACTCAGTCTCCAGCCTCCCT 338 208B-408L DIQMTQSPASLSASVGETVTITCATCTGCATCTGTGGGAGAAACTGTCACCATCA RASGNIHTYLAWYQQKQGESPQLCATGTCGAGCAAGTGGGAATATTCACACTTAT LVYNANTLADGVPSRFSGSGSGTTTAGCATGGTATCAGCAGAAACAGGGAAAATC QFSLKINSLQPDDEGSYYCQHFWTCCTCAGCTCCTGGTCTACAATGCAAACACCT SAPWTFGGGTQLEIKRTGGCAGATGGTGTGCCATCAAGGTTCAGTGGC AGTGGATCAGGAACACAATTTTCTCTCAAGATCAACAGTCTGCAGCCTGACGATTTTGGGAGTT ATTACTGTCAACATTTTTGGAGTGCTCCGTGGACGTTCGGTGGAGGCACCCAGCTGGAAATCAA ACGGGCTGATGCTGC 208A-207L 339GATATTGTGTTAACTCAGTCTCCAGCCACCCT 340 208A-207L DIVLTQSPATLSVTPGDRVSLSC208B-826L GTCTGTGACTCCAGGAGATAGAGTCAGTCTTT 208B-826LRASQRIYNYLHWYQQESHESPRL CCTGCAGGGCCAGTCAAAGAATTTACAACTACLTHYASQSISGIPSRFSGSGSGT CTACACTGGTATCAACAAAAATCACATGAGTCDFILTINSVETEDFGMYFCQQSN TCCAAGGCTTCTCACCAAGTATGCTTCCCAGTSWPLTFGAGTKLELRR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGCTCAGGGACAGATTTCATTCTCACTAT CAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCTCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGACGGGCTGATGCTGC 208B-395L 341 GATATTGTGTTAACTCAGTCTCCAGCCACCCT 342208B-395L DIVLTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCAGTCTTTRASQRIYNYLHWYQQESHESPRL CCTGCAGGGCCAGTCAAAGAATTTACAACTACLIKYASQSISGIPSRFSGSGSGT CTACACTGGTATCAACAAAAATCACATGAGTCDFILTINSVETEDFGMYFCQQSN TCCAAGGCTTCTCATCAAGTATGCTTCCCAGTSWPLTFGAGTKLELRR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGCTCAGGGACAGATTTCATTCTCACTAT CAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCTCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGACGGGCTGATGCTGC 208B-517L 343 GACATTGTGATGACTCAGTCTCCAGCCACCCT 344208B-517L DIVMTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCTCTCTTTRASQSISDYLHWYQQESHESPRL CCTGCAGGGCCAGCCAGAGTATTAGCGACTACLIKYASQSISGIPSRFRGSGSGS TTACACTGGTATCAACAAAAATCACATGAGTCHFTLSINSVEPEDVGVYYCQNGH TCCAAGGCTTCTCATCAAATATGCTTCCCAATSFPWTFGGGTKLEIKR CCATCTCTGGGATCCCCTCCAGGTTCCGTGGCAGTGGATCAGGGTCACATTTCACTCTCAGTAT CAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAATGGTCACAGTTTTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGC 208B-822L 345 GACATTGTGATGACTCAGTCTCCAGCCACCCT 346208B-822L DIVMTQSPATLSVTPGDRVSLSC GTCTGTGACTCCAGGAGATAGAGTCTCTCTTTRASQTISDYLHWYQQESHESPRL CCTGCAGGGCCAGCCAGACTATTAGCGACTACLIKYASQSISGIPSRFSGSGSGS TTACACTGGTATCAACAAAAATCGCATGAGTCHFTLSINSVEPEDVGVYYCQNGH TCCAAGGCTTCTCATCAAATATGCTTCCCAATSFPWTFGGGTKLEIKR CCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCACATTTCACTCTCAGTAT CAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAATGGTCACAGTTTTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGC 208A-210L 347 GAGATTGTGCTCACTCAGTCTCCAGCCATCAC 348208A-210L EIVLTQSPAITAASLGQNVTITC AGCTGCATCTCTGGGGCAAAACGTCACCATCARASSSVSYMHWYRQESGTSPQLW CCTGCAGAGCCAGCTCAAGTGTAAGTTACATGIYEISRRASGVPARFRASGSGTS CATTGGTACCGGCAGAAGTCCGGCACCTCCCCYSLTISSMEAEDAAIYYCQQWNY CCAACTATGGATTTATGAGATATCCAGACGGG PLTFGAGTKLEVKRCTTCTGGAGTCCCAGCTCGCTTCCGTGCCAGT GGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATT ACTGCCAGCAGTGGAATTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGGTGAAACGGGC TGATGCTGC 208B-547L 349GAAGTTGTGCTCACTCAGTCTCCAGCCATCAC 350 208B-547L EVVLTQSPAITAASLGQKVTITCAGCTGCATCTCTGGGGCAAAAGGTCACCATCA RASSSVSYMHWYRQESGTSPQPWCCTGCAGAGCCAGCTCAAGTGTAAGTTACATG IYEISTLASGVPTRFRASGSGTSCACTGGTACCGGCAGAAGTCAGGCACCTCCCC YSLTISSMEAEDAAIYYCQQWNYCCAGCCATGGATTTATGAAATATCCACACTGG PLTFGAGTKLELKRCTTCTGGAGTCCCAACTCGCTTCCGTGCCAGT GGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATT ACTGCCAGCAGTGGAATTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAACTGAAACGGGC TGATGCTGC 208A-638L 351CAAATTGTTCTCACCCAGTCTCCAACAATCAT 352 208A-638L QIVLTQSPTIMSASLGERVTMTCGTCTGCATCTCTAGGGGAACGGGTCACCATGA TASSSVSSSYLHWFQQFPGSSPECCTGCACTGCCAGCTCAAGTGTGAGTTCCAGT LWIYSTSNLASGVPPRFSGSGSGTACTTGCACTGGTTCCAGCAGAAGCCAGGATC TSYSLSISSVEAEDVATYYCLQFCTCCCCCAAACTCTGGATTTATAGCACATCCA HRSPWTFEGGGAKLEIKRACCTGGCTTCTGGAGTCCCACCTCGCTTCAGT GGCAGTGGGTCTGGGACCTCTTACTCTCTCTCAATCAGCAGCGTGGAGGCTGAAGATGTTGCCA CTTATTACTGCCTCCAGTTTCATCGTTCCCCGTGGACGTTCGGTGGAGGCGCCAAGTTGGAAAT CAAACGGGCTGATGCTGC 208B-515L 353CAAATTGTTTTCACCCAGTATCCAGCAATAAT 354 208B-515L QIVFTQYPAIMSASLGERVTMTCGTCTGCATCTCTAGGGGAACGGGTCACCATGA TASSSVTSSYLHWFQQFPGSSPKCCTGCACAGCCAGCTCAAGTGTAACTTCCAGT LWIYSTSNPGSGVPARFSGRGSGTACTTGCACTGGTTCCAGCAGAAGCCAGGATC TSYSLSISSMEAEDAATYYCLQFCTCCCCCAAACTCTGGATTTATAGCACGTCCA HRSPWTFGGGTKLEIRRACCCGGGTTCTGGAGTCCCAGCTCGCTTCAGT GGCAGAGGATCTGGGACCTCTTACTCTCTCTCAATCAGCAGCATGGAGGCTGAAGATGCTGCCA CTTATTACTGCCTCCAGTTTCATCGTTCCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAAT CAGACGGGCTGATGCTGC 208A-877L 355CAAATTGTTCTCACCCAGTCTCCAGCAATCAT 356 208A-877L QIVLTQSPAIMSVSPGEKVSMTCGTCTGTATCTCCAGGGGAGAAGGTCTCCATGA SASSSVTYMHWYQQESGTSPERWCCTGCAGTGCCAGCTCAAGTGTCACTTACATG IYDTSELASGVPARFSGSGSGTTCACTGGTATCAGCAGAAGTCAGGCACCTCCCC YSLTISSMEAEDAATYYCQQWSNCAAAAGATGGATTTATGACACATCCGAGCTGG KPLTFGAGTKLELKRCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGT GGGTCTGGGACCACTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGCAGTGGAGTAATAAACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACG GGCTGATGCTGC 208B-174L 357CAAAATGTTCTCACCCAGTCTCCAGCAATCAT 358 208B-174L QNVLTQSPAIMSASPGEKVTMSCGTCTGCATCTCCAGGGGAGAAGGTCACCATGT SASSSVTYMEWYQLEPESSPRLLCCTGCAGTGCCAGCTCAAGTGTCACTTACATG IYDTSNLASGVPVRFSGSGSGTSTTCTGGTACCAGCTGAAGCCAGAATCCTCCCC YSLTISRMEAEDAATYYCQEWSSCAGACTCCTGATTTATGACACATCCAATTTGG YPLTFGAGTKLDLKRCTTCTGGCGTCCCTGTTCGCTTCAGTGGCAGT GGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGTATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGGAGTGGAGTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGACCTGAAACG GGCTGATGCTGC 208B-612L 359CAAATTGTTCTCACCCAGTCTCCAACACTCAT 360 208B-612L QIVLTQSPTLMSASPGEKVTMTCGTCTGCATCGCCAGGAGAAAAGGTCACCATGA SASSTVTYIYWYQQFPGSSPRLWCCTGCAGTGCCAGCTCAACTGTGACTTACATT MYDTFNLVSGVPARFSGSRSGTSTACTGGTACCAACAGAAGCCCGGCTCCTCCCC YFLTISSMEGEDAATYYCQQYSDCAGACTCTGGATGTATGACACATTCAACCTGG SPYTFGGGTKLEIKRTTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGT AGGTCTGGGACCTCTTATTTTCTCACAATCAGTAGCATGGAGGGTGAAGATGCTGCCACTTATT ACTGCCAACAGTACAGTGATTCCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAACG GGCTGATGCTGC 208B-911L 361CAAATTGTTCTCACCCAGTCTCCAGAGATCAT 362 208B-911L QIVLTQSPEIMSASPGEKVTITCGTCTGCATCTCCAGGGGAGAAGGTCACCATAA SASSSVSFMYWFQQFPGTSPELWCCTGCAGTGCCAGCTCAAGTGTAAGTTTCATG IYITSNLASGVPTRFSGSGSGTSTATTGGTTCCAGCAGAAGCCAGGCACTTCTCC YSLTISRMEAEDAATYYCQQRSSCAAACTCTGGATTTATATCACATCCAACCTGG FPYTFGGGTKLEMKRCTTCTGGAGTCCCTACTCGCTTCAGTGGCAGT GGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATT ACTGCCAGCAAAGGAGTAGTTTCCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATGAAACG GGCTGATGCTGC 208A-422L 363GACATTGTGCTGACCCAATCTCCAGCTTCTTT 364 208A-422L DIVLTQSPASLAVSLGQRAIISC208A-442L GGCTGTGTCTCTAGGGCAGAGGGCCATCATCT 208A-442LKASQSVSFAGTNLMHWYQQFPGQ CCTGCAAGGCCAGCCAAAGTGTCAGTTTTGCTQPELLITRASNLETGVPTRFSGS GGTACTAATTTAATGCACTGGTACCAACAGAAGSRTDFTLNIHPVEEDDAATYYC ACCAGGACAGCAACCCAAACTCCTCATCTATCQQSREYYTFGGGTKLEIKR GTGCATCCAACCTAGAAACTGGGGTTCCTACCAGGTTTAGTGGCAGTGGGTCTAGGACAGACTT CACCCTCAATATCCATCCTGTGGAGGAAGATGATGCTGCAACCTATTACTGTCAGCAAAGTAGG GAATATTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGC 208A-967L 365 GACATTGTGCTGACCCAATCTCCAGCTTCTTT366 208A-967L DIVLTQSPASLAGSLGERAPISC GGCTGGGTCTCTAGGGAAAAGGGCCCCCATCTKASESVNEFGTNLIHWYQQFPGQ CCTGCAAAGCCAGCGAAAGTGTCAATTTTTTTPPELLIYHASNLKTGVPARFRGR GGTACTAATTTAATACACTGGTACCAACAAAAGSKTNFPLPIDPVEENDVAITYC ACCAGGACAGCCCCCCAAACTCCTCATCTATCLQNRKIPLTFGVGTKLELKR ATGCATCCAACCTAAAAACTGGAGTCCCTGCCAGGTTCAGGGGCAGGGGGTCTAAAACAAACTT CCCCCTCCCCATTGATCCTGTGGAGGAAAATGATGTTGCAATCTATTACTGTCTGCAAAATAGG AAAATTCCTCTCACGTTCGGGGTTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGC 208B-178L 367GACATTGTGCTGACCCAATCTCCAGCTTCTTT 368 208B-178L DIVLTQSPASLAVSLGQRATISCGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT RASESVDNYGISFMHWYQQFPGQCCTGCAGAGCCAGCGAAAGTGTTGATAATTAT PPELLITRASNLESGIPARFSGSGGCATTAGTTTTATGCACTGGTACCAGCAGAA GSRTDFTLTINPVETDDVATYYCACCAGGACAGCCACCCAAACTCCTCATCTATC QQSNEDPFTFGSGAKLEIERGTGCATCCAACCTAGAATCTGGGATCCCTGCC AGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGACTGATG ATGTTGCAACCTATTACTGTCAGCAAAGTAATAAGGATCCATTCACGTTCGGCTCGGGGGCAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-222L369 GACATTGTGGTGACACAGTCTCCTGCTTCCTT 370 208A-222LDIVVTQSPASLAVSLGQRATISC 208A-605L AGCTGTATCTCTGGGGCAGAGGGCCACCATCT208A-605L RASQSVSTSRYSYLHWYQQFPGQ 208B-560LCATGCAGGGCCAGCCAAAGTGTCAGTACATCT 208B-560L PPELLIKYASNLESGVPARFSGSAGATATAGTTATCTGCACTGGTACCAACAGAA GSGTDFTLNIHPVGEEDTATYYCACCAGGACAACCTCCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKRATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAATATCCATCCTGTGGGGGAGGAGG ATACTGCAACATATTACTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-830L371 GACATTGTACTGACACAGTCTCCTGCTTCCTT 372 208A-830LDIVLTQSPASLAVSLGQRATISC AGCTGTATCTCTGGGGCAGAGGGCCACCATCTRSSQSVSTSRYSYLHWYQQFPGQ CATGCAGGTCCAGCCAAAGTGTCAGTACATCTPPELLIKYASNLESGVPARFSGS AGATATAGTTATTTGCACTGGTACCAACAGAAGSGTDFTLNIHPVGEEDPATYYC ACCAGGACAACCTCCCAAACTCCTCATCAAGTQHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGGGGAGGAGGATCCTGCAACATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGC 208A-557L 373GACATTGCCCTGACACAGTCTCCTGCTTCGTT 374 208A-557L DIALTQSPASLAVSLGQRATISCAGCTGTATCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGQCATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGSAGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVGEEDTATYYCACCAGGACAACCACCCGAACTCCTCATCAAGT QHSWDFPFTFGSGTKLEIKRATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGGGGAGGAGG ATACTGCAACATATTACTGTCAGCACAGTTGGGATTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-133L375 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 376 208A-133LDIVLTQSPASLAVSLGQRATISC 208B-556L AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT208B-556L RASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCTPPELLIKYASNLESGVPARFSGS AGGTATAGCTACATGCACTGGTACCAACAGAAGSGTDFTLNIHPVAEEDTATYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGTQHSWEFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGC 208B- 377 GACATTGTGCTGACACAGTCTCTTGCTTCCTT378 208B-1070L DIVLTQSLASLAVSLGQRATISC 1070LAGCTGTTTCTCTGGGGCAGAGGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGQCATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLECGVRARFSGSAGGTATAGTTATATGCACTGGTACCAACAGAA GCGTDFTLNIHPVEEEDTAAYYCACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKRATGCATCCAACCTTGAATGTGGGGTCCGTGCC AGGTTCAGTGGCAGTGGGTGTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA ATTGGAAATAAAACGGGCTGATGCTGC 208A-159L379 GACATTGTACTGACACAGTCTCCTGCTTCCTT 380 208A-159LDIVLTQSPASLAVSLGQRATISC AGCTGTTTCTCTGGGGCAGAGGGCCACCATCTRASQSVSTSRYSYVHWYQQFPGQ CCTGCAGGGCCAGCCAAAGTGTCAGTACATCTPPELLIKYAANLESGVPARFSGS AGGTATAGTTATGTGCACTGGTATCAACAGAAGSGTDFTLNIHPVAEEDAAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGTQHSWEFPFTFGSGTKLEIKR ATGCAGCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGGATGCTGCAGCATATTACTGTCAGCACAGTTGG GAGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGC 208A-741L 381GACATTGTGCTGACACAGTCTCCTGCTTCCTT 382 208A-741L DIVLTQSPASLAVSLGQRTTISCAGCTGTTTCTCTGGGGCAGAGGACCACCATCT GASQSVSTSRFSYMHWYQQFPGQCATGCGGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGSAGGTTTAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYCACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKRATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-334L383 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 384 208A-334LDIVLTQSPASLAVSLGQRATISC AGCTGTTTCTCTGGGGCAGAGGGCCACCATCTRASQSVSTSRYSYMHWYQQFPGQ CATGCAGGGCCAGCCAAAGTGTCAGTACATCTPPELLIKYASNLESGVPARFSGS AGGTATAGTTATATGCACTGGTACCAACAGAAGSGTDFTLNIHPVAEEDTAAYYC ACCAGGACAGCCACCCAAACTCCTCATCAAGTQHSWGFPFTFGSGTKLEIKR ATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACCCTCAACATCCATCCTGTGGCGGAGGAGGATACTGCAGCATATTACTGTCAGCACAGTTGG GGGTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAGCGGGCTGATGCTGC 208B-251L 385GACATTGTGCTGACACAGTCTCCTGCTTCCTT 386 208B-251L DIVLTQSPASLAVSLGEKATISCAGCTGTATCTTTGGGAAAAAAGGCCACCATCT RASQSVSTSRYSYMHWYQQFPGHCATGCAGGGCCAGCCAAAGTGTCAGTACATCT PPELLIKYASNLESGVPARFSGSAGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYCACCAGGACACCCACCCAAACTCCTCATCAAAT QHSWEFPFTFGSGTKLEIKRATGCATCCAACCTAGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAACGGGCTGATGCTGC 208A-1101387 GACATTGTGCTGACACAGTCTCCTGCTTCCTT 388 208A-1101DIVLTQSPASLAVSLGQRATISC 208A-126 AGCTGTTTCTCTGGGGCAGAGGGCCACCATCT208A-126 RASQSVSTSRYSYMHWYQQKPGQ 208A-9201CATGCAGGGCCAGCCAAAGTGTCAGTACATCT 208A-9201 PPKLLIKYASNLESGVPARFSGSAGGTATAGTTATATGCACTGGTACCAACAGAA GSGTDFTLNIHPVAEEDTAAYYCACCAGGACAGCCACCCAAACTCCTCATCAAGT QHSWEFPFTFGSGTKLEIKRATGCATCCAACCTTGAATCTGGGGTCCCTGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGCGGAGGAGG ATACTGCAGCATATTACTGTCAGCACAGTTGGGAGTTTCCATTCACGTTCGGCTCGGGGACAAA ATTGGAAATAAAACGGGCTGATGCTGC 208A- 389AGATCTAGTCAGAGCCTTGAACATAGTAATGG 390 208A- RSSQSLEHSNGNTYLE 692CDRL1AAACACCTATTTAGAG 692CDRL1 208A- 391 AGATCTAGTCAGAGCCTTGTACATAGTAATGG 392208A- RSSQSLVHSNGNTYLE 352CDRL1 AAACACCTATTTAGAG 352CDRL1 208A- 393AGATCTAGTCAGAGCCTTGTACATAGTAATGG 394 208A- RSSQSLLHSNGNTYLE 983CDRL1AAACACCTATTTAGAG 983CDRL1 208B- 208B- 471CDRL1 471CDRL1 208B- 395AGATCTAGTCAGAGCATTGTACACAGTAATGG 396 208B- RSSQSIVHSNGNTYLE 1024CDRL1AAACACCTATTTAGAG 1024CDRL1 208A- 397 AGATCTAGTCAGAGCCTTGAACATACTAATGG398 208A- RSSQSLEHTNGNTYLE 134CDRL1 AAACACCTATTTAGAG 134CDRL1 208B- 399AGATCTAGTCAGAGCCTTGTACATAATAATGG 400 208B- RSSQSLVHNNGNTYLE 281CDRL1AAACACCTATTTAGAA 281CDRL1 208B- 401 AGGTCTAGTAAGAGTCTCCTACATAGTAATGG 402208B- RSSKSLLHSNGITYLY 327CDRL1 CATCACTTATTTGTAT 327CDRL1 208B- 403AGATCTAGTCAGACCCTTCTACACAGTGATGG 404 208B- KESQTLLHRDGNPFLL 862CDRL1AGACACCTATTTACAT 862CDRL1 208B- 405 AAGTCAAGTCAGAGCCTCTTATTTACTAATGG 406208B- KSSQSLLFTNGKTYLN 589CDRL1 AAAAACCTATTTAAAT 589CDRL1 208B- 208B-1096CDRL1 1096CDRL1 208B- 407 AAGTCAAGTCAGAGCCTCTTATATACTAATGG 408 208B-KSSQSLLYTNGKTYLN 189CDRL1 AAAGACCTATTTGAAT 189CDRL1 208A- 409AAGGCCAGTCAGAATGTGAGTCCTGCTGTAGC 410 208A- KASQNVSPAVA 874CDRL1 C874CDRL1 208B- 411 AAGGCCAGTCAGAATGTGGGTACTGCTGTAGC 412 208B-KASQNVGTAVA 353CDRL1 C 353CDRL1 208B- 413AAGGCCAGTCAGGATGTGGGTACTGCTGTAGC 414 208B- KASQDVGTAVA 793CDRL1 C793CDRL1 208B- 415 CGGGCAAGTCAGGACATTGGTGGTAGCATAAA 416 208B-RASQDIGGSIN 672CDRL1 C 672CDRL1 208B- 417CGAGCAAGTGGGAATATTCACACTTATTTAGC 418 208B- RASGNIHTYLA 408CDRL1 A408CDRL1 208A- 419 AGGGCCAGTCAAAGAATTTACAACTACCTACA 420 208A-RASQRIYNYLH 207CDRL1 C 207CDRL1 208B- 208B- 826CDRL1 826CDRL1 208B-208B- 395CDRL1 395CDRL1 208B- 421 AGGGCCAGCCAGAGTATTAGCGACTACTTACA 422208B- RASQSISDYLH 517CDRL1 C 517CDRL1 208B- 423AGGGCCAGCCAGACTATTAGCGACTACTTACA 424 208B- RASQTISDYLH 822CDRL1 C822CDRL1 208A- 425 AGAGCCAGCTCAAGTGTAAGTTACATGCAT 426 208A- RASSSVSYMH210CDRL1 210CDRL1 208B- 427 AGAGCCAGCTCAAGTGTAAGTTACATGCAT 428 208B-RASSSVSYMH 547CDRL1 547CDRL1 208A- 429 ACTGCCAGCTCAAGTGTGAGTTCCAGTTACTT430 208A- TASSSVSSSYLH 638CDRL1 GCAC 638CDRL1 208B- 431ACAGCCAGCTCAAGTGTAACTTCCAGTTACTT 432 208B- TASSSVTSSYLH 515CDRL1 GCAC515CDRL1 208A- 433 AGTGCCAGCTCAAGTGTCACTTACATGCAC 434 208A- SASSSVTYMH877CDRL1 877CDRL1 208B- 435 AGTGCCAGCTCAAGTGTCACTTACATGTTC 436 208B-SASSSVTYMF 174CDRL1 174CDRL1 208B- 437 AGTGCCAGCTCAACTGTGACTTACATTTAC438 208B- SASSTVTYIY 612CDRL1 612CDRL1 208B- 439AGTGCCAGCTCAAGTGTAAGTTTCATGTAT 440 208B- SASSSVSFMY 911CDRL1 911CDRL1208A- 441 AAGGCCAGCCAAAGTGTCAGTTTTGCTGGTAC 442 208A- KASQSVSFAGTNLMH422CDRL1 TAATTTAATGCAC 422CDRL1 208A- 208A- 442CDRL1 442CDRL1 208A- 443AAAGCCAGCGAAAGTGTCAATTTTTTTGGTAC 444 208A- KASESVNFFGTNLIH 967CDRL1TAATTTAATACAC 967CDRL1 208B- 445 AGAGCCAGCGAAAGTGTTGATAATTATGGCAT 446208B- RASESVDNYGISFMH 178CDRL1 TAGTTTTATGCAC 178CDRL1 208A- 447AGGGCCAGCCAAAGTGTCAGTACATCTAGATA 448 208A- RASQSVSTSRYSYLH 222CDRL1TAGTTATCTGCAC 222CDRL1 208A- 208A- 605CDRL1 605CDRL1 208B- 208B-560CDRL1 560CDRL1 208A- 449 AGGGCCAGCCAAAGTGTCAGTACATCTAGGTA 450 208A-RASQSVSTSRYSYMH 557CDRL1 TAGTTATATGCAC 557CDRL1 208A- 208A- 133CDRL1133CDRL1 208B- 208B- 556CDRL1 556CDRL1 208B- 208B- 1070CDRL1 1070CDRL1208B- 208B- 251CDRL1 251CDRL1 208A- 208A- 110CDRL1 110CDRL1 208A- 208A-126CDRL1 126CDRL1 208A- 208A- 920CDRL1 920CDRL1 208A- 208A- 334CDRL1334CDRL1 208A- 451 GGGGCCAGCCAAAGTGTCAGTACATCTAGGTT 452 208A-GASQSVSTSRFSYMH 741CDRL1 TAGTTATATGCAC 741CDRL1 208A- 453AGGTCCAGCCAAAGTGTCAGTACATCTAGATA 454 208A- RSSQSVSTSRYSYLH 830CDRL1TAGTTATTTGCAC 830CDRL1 208A- 455 AGGGCCAGCCAAAGTGTCAGTACATCTAGGTT 456208A- RASQSVSTSRFSYLH 1064CDRL1 TAGTTATCTGCAC 1064CDRL1 208A- 457AGGGCCAGCCAAAGTGTCAGTACATCTAGGTA 458 208A- RASQSVSTSRYSYVH 159CDRL1TAGTTATGTGCACTGG 159CDRL1 208A- 459 AGGGCCAGCCAAAGTGTCAGTACATCCAGGTT 460208A- RASQSVSTSRFSYVH 293CDRL1 TAGTTATGTGCAC 293CDRL1 208B- 461AGGGCCAGCCAAAGTCTCAGTACATCTAGGTT 462 208B- RASQSLSTSRFSYVH 1094CDRL1TAGCTATGTGCAC 1094CDRL1 208A- 463 AAAGTTTCCAGCCGATTTTCT 464 208A-KVSSRFS 692CDRL2 692CDRL2 208A- 208A- 134CDRL2 134CDRL2 208B- 465AAAGTTTCCAACCGATTTTCT 466 208B- KVSNRFS 281CDRL2 281CDRL2 208B- 467AAACTTTCCAACCGATTTTCT 468 208B- KLSNRFF 862CDRL2 862CDRL2 208A- 469AATGTTTCCAACCGATTTTCT 470 208A- NVSNRFS 352CDRL2 352CDRL2 208A- 208A-983CDRL2 983CDRL2 208B- 208B- 471CDRL2 471CDRL2 208B- 471AGAGTTTCCAACCGATTTTCT 472 208B- RVSNRFS 1024CDRL2 1024CDRL2 208B- 473CAGATGTCCAAGATTGCCTCA 474 208B- QMSKIAS 327CDRL2 327CDRL2 208B- 475CTGCTGTCTAAATTGGACTCT 476 208B- LLSKLDS 589CDRL2 589CDRL2 208B- 208B-1096CDRL2 1096CDRL2 208B- 477 CTGGTGTCAAAATTGGACTCT 478 208B- LVSELDS189CDRL2 189CDRL2 208A- 479 TCGGCATCCTCCCGATACACT 480 208A- SASSRYT874CDRL2 874CDRL2 208B- 481 TCAGCATCCAATCGGTACACT 482 208B- SASNRYT353CDRL2 353CDRL2 208B- 483 TGGGCATCCACCCGGCACACT 484 208B- WASTRHT793CDRL2 793CDRL2 208B- 485 GCCACATCCAGTTTAGATTCT 486 208B- ATSSLDS672CDRL2 672CDRL2 208B- 487 AATGCAAACACCTTGGCAGAT 488 208B- NANTLAD408CDRL2 408CDRL2 208A- 489 GCTTCCCAGTCCATCTCTGGG 490 208A- ASQSISG207CDRL2 207CDRL2 208B- 208B- 826CDRL2 826CDRL2 208B- 208B- 395CDRL2395CDRL2 208B- 208B- 517CDRL2 517CDRL2 208B- 491 TATGCTTCCCAATCCATCTCT492 208B- YASQSIS 822CDRL2 822CDRL2 208A- 493 GAGATATCCAGACGGGCTTCT 494208A- EISRRAS 210CDRL2 210CDRL2 208B- 495 GAAATATCCACACTGGCTTCT 496208B- EISTLAS 547CDRL2 547CDRL2 208A- 497 AGCACATCCAACCTGGCTTCT 498208A- STSNLAS 638CDRL2 638CDRL2 208B- 499 AGCACGTCCAACCCGGGTTCT 500208B- STSNPGS 515CDRL2 515CDRL2 208A- 501 GACACATCCGAGCTGGCTTCT 502208A- DTSELAS 877CDRL2 877CDRL2 208B- 503 GACACATCCAATTTGGCTTCT 504208B- DTSNLAS 174CDRL2 174CDRL2 208B- 505 GACACATTCAACCTGGTTTCT 506208B- DTFNLVS 612CDRL2 612CDRL2 208B- 507 ATCACATCCAACCTGGCTTCT 508208B- ITSNLAS 911CDRL2 911CDRL2 208A- 509 CGTGCATCCAACCTAGAAACT 510208A- RASNLET 422CDRL2 422CDRL2 208A- 208A- 442CDRL2 442CDRL2 208A- 511CATGCATCCAACCTAAAAACT 512 208A- HASNLET 967CDRL2 967CDRL2 208B- 513CGTGCATCCAACCTAGAATCT 514 208B- RASNLES 178CDRL2 178CDRL2 208A- 515TATGCATCCAACCTAGAATCT 516 208A- YASNLES 222CDRL2 222CDRL2 208A- 208A-605CDRL2 605CDRL2 208B- 208B- 560CDRL2 560CDRL2 208A- 208A- 557CDRL2557CDRL2 208A- 208A- 133CDRL2 133CDRL2 208B- 208B- 556CDRL2 556CDRL2208B- 208B- 251CDRL2 251CDRL2 208A- 208A- 110CDRL2 110CDRL2 208A- 208A-126CDRL2 126CDRL2 208A- 208A- 920CDRL2 920CDRL2 208A- 208A- 334CDRL2334CDRL2 208A- 208A- 741CDRL2 741CDRL2 208A- 208A- 830CDRL2 830CDRL2208A- 208A- 1064CDRL2 1064CDRL2 208A- 208A- 159CDRL2 159CDRL2 208A-208A- 293CDRL2 293CDRL2 208B- 208B- 1094CDRL2 1094CDRL2 208B- 517TATGCATCCAACCTTGAATGT 518 208B- YASNLEC 1070CDRL2 1070CDRL2 208A- 519TTTCAAGGTTCACATGTTCCATTCACG 520 208A- FQGSHVPFT 692CDRL3 692CDRL3 208A-208A- 983CDRL3 983CDRL3 208B- 208B- 281CDRL3 281CDRL3 208A- 208A-134CDRL3 134CDRL3 208A- 521 TTTCAAGGTTCACATGTTCCACTCACG 522 208A-FQGSHVPLT 352CDRL3 352CDRL3 208B- 208B- 471CDRL3 471CDRL3 208B- 523TTTCAAGGTTCACATGTTCCGTGGACG 524 208B- FQGSHVPWTF 1024CDRL3 1024CDRL3208B- 525 GCTCAAAATCTAGAACTTCCGTGGACG 526 208B- AQNLELPWT 327CDRL3327CDRL3 208B- 527 TCTCAAAGTACACATGTTCCGTACACG 528 208B- SQSTHVPYT862CDRL3 862CDRL3 208B- 529 TTGCAGAGTACATATTTTCCTCTCACG 530 208B-LQSTYFPLT 589CDRL3 589CDRL3 208B- 208B- 1096CDRL3 1096CDRL3 208B- 531TTGCAGAGTATACATTTTCCGTACACG 532 208B- LQSIHFPYT 189CDRL3 189CDRL3 208B-533 CTACAATATGCTAGTTCTCCTCCGACG 534 208B- LQYASSPPT 672CDRL3 672CDRL3208A- 535 CTCCAGTTTCATCGTTCCCCGTGGACG 536 208A- LQFHRSPWT 638CDRL3638CDRL3 208B- 208B- 515CDRL3 515CDRL3 208A- 537CTGCAAAATAGGAAAATTCCTCTCACG 538 208A- LQNRKIPLT 967CDRL3 967CDRL3 208A-539 CAGCAACATTTTAGTACTCCGTGGACG 540 208A- QQHFSTPWT 874CDRL3 874CDRL3208B- 541 CAGCAATATAGCACCTATCCTCTCACG 542 208B- QQYSTYPLT 353CDRL3353CDRL3 208B- 543 CAGCAATATAGCAACTATCTCACG 544 208B- QQYSNYLT 793CDRL3793CDRL3 208A- 545 CAACAGAGTAACAGCTGGCCTCTCACG 546 208A- QQSNSWPLT207CDRL3 207CDRL3 208B- 208B- 826CDRL3 826CDRL3 208B- 208B- 395CDRL3395CDRL3 208A- 547 CAGCAGTGGAATTATCCTCTCACG 548 208A- QQWNYPLT 210CDRL3210CDRL3 208B- 208B- 547CDRL3 547CDRL3 208A- 549CAGCAGTGGAGTAATAAACCGCTCACG 550 208A- QQWSNIKPLT 877CDRL3 877CDRL3 208B-551 CAACAGTACAGTGATTCCCCGTACACG 552 208B- QQYSDSPYT 612CDRL3 612CDRL3208B- 553 CAGCAAAGGAGTAGTTTCCCGTACACG 554 208B- QQRSSFPYT 911CDRL3911CDRL3 208A- 555 CAGCAAAGTAGGGAATATTACACG 556 208A- QQSREYYT 422CDRL3422CDRL3 208A- 208A- 442CDRL3 442CDRL3 208B- 557CAGCAAAGTAATAAGGATCCATTCACG 558 208B- QQSNEDPFT 178CDRL3 178CDRL3 208B-559 CAAAATGGTCACAGTTTTCCGTGGACG 560 208B- QNGHSFPWT 517CDRL3 517CDRL3208B- 208B- 822CDRL3 822CDRL3 208B- 561 CAACATTTTTGGAGTGCTCCGTGGACG 562208B- QHFWSAPWT 408CDRL3 408CDRL3 208B- 563 CAGGAGTGGAGTAGTTACCCACTCACG564 208B- QEWSSYPLT 174CDRL3 174CDRL3 208A- 565CAGCACAGTTGGGAGTTTCCATTCACG 566 208A- QHSWEFPFT 222CDRL3 222CDRL3 208A-208A- 605CDRL3 605CDRL3 208B- 208B- 560CDRL3 560CDRL3 208A- 208A-133CDRL3 133CDRL3 208B- 208B- 556CDRL3 556CDRL3 208B- 208B- 1070CDRL31070CDRL3 208B- 208B- 251CDRL3 251CDRL3 208A- 208A- 110CDRL3 110CDRL3208A- 208A- 126CDRL3 126CDRL3 208A- 208A- 920CDRL3 920CDRL3 208A- 208A-741CDRL3 741CDRL3 208A- 208A- 830CDRL3 830CDRL3 208A- 208A- 1064CDRL31064CDRL3 208A- 208A- 159CDRL3 159CDRL3 208A- 208A- 293CDRL3 293CDRL3208B- 208B- 1094CDRL3 1094CDRL3 208A- 567 CAGCACAGTTGGGATTTTCCATTCACG568 208A- QHSWDFPFT 557CDRL3 557CDRL3 208A- 569CAGCACAGTTGGGATTTTCCATTCACG 570 208A- QHSWGFPFT 334CDRL3 334CDRL3HCV core 571 ATGTCTACCAACCCGAAACCGCAGAAAAAAAA 572 HCV core 1-MSTNPKPQKKNKRNTNRRPQDVK 1-169 CAAACGTAACACCAACCGTCGTCCGCAGGACG 169 aminoFPGGGQIVGGVYLLPRRGPRLGV amino TTAAATTCCCGGGTGGTGGTCAGATCGTTGGT acidRATRKTSERSQPRGRRQPIPKAR acid GGTGTTTACCTGCTGCCGCGTCGTGGTCCGCG sequenceRPEGRTWAQPGYPWPLYGNEGCG sequence TCTGGGTGTTCGTGCTACGCGTAAAACCTCTGWAGWLLSPRGSRPSWGPTDPRRR AACGTTCTCAGCCGCGTGGGCGTCGTCAGCCGSRNLGKVIDTLTCGFADLMGYIP ATCCCGAAAGCTCGTCGTCCGGAAGGTCGTACLVGAPLGGAARALAHGVRVLEDG CTGGGCTCAGCCGGGTTACCCGTGGCCGCTGT VNYATGNLACGGTAACGAAGGTTGCGGTTGGGCTGGTTGG CTGCTGTCTCCGCGTGGATCTCGTCCGTCTTGGGGTCCGACCGACCCGCGTCGTCGTTCTCGTA ACCTTGGTAAAGTTATCGATACCCTGACCTGCGGTTTCGCTGACCTGATGGGTTACATACCGCT GGTTGGAGCTCCGCTGGGTGGTGCTGCTCGTGCTCTGGCGCATGGCGTGCGTGTTCTGGAAGAT GGCGTCAACTATGCCACCGGTAATCTG 573HCV 134-154 MGYIPLVGAPLGGAARALAHG Peptide 1 574 HCV 141-161GAPLGGAARALAHGVRVLEDG Peptide 2 575 HCV 151-171 LAHGVRVLEDGVNYATGNLPGPeptide 3 576 ALRZ-8 MGYIPLVGAPLGGAARALAHGVR VLEDGVNYATGNLPGC 577 ALRZ-9MGYIPLVGAPLGGAARALAHGVR VLEDGVNYATGNLPGQYIKANSK FIGITEL

1.-16. (canceled)
 17. A system comprising: (a) a first antibody, orantigen-binding portion thereof, which comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences of SEQ ID NO: 588, SEQ ID NO: 589, and SEQID NO: 590, respectively, and light chain CDR1, CDR2, and CDR3 aminoacid sequences of SEQ ID NO: 585, SEQ ID NO: 586, and SEQ ID NO: 587;and (b) a second antibody, or antigen-binding portion thereof, whichspecifically binds to the amino acid sequence of SEQ ID NO: 574 in thelipid binding domain of HCV core protein. 18.-21. (canceled)
 22. Thesystem of claim 17, wherein the first and/or second antibody, or theantigen-binding portion thereof, comprises a detectable label attachedthereto.
 23. A kit comprising: (a) a first antibody, or antigen-bindingportion thereof, which comprises heavy chain CDR1, CDR2, and CDR3 aminoacid sequences of SEQ ID NO: 588, SEQ ID NO: 589, and SEQ ID NO: 590,respectively, and light chain CDR1, CDR2, and CDR3 amino acid sequencesof SEQ ID NO: 585, SEQ ID NO: 586, and SEQ ID NO: 587; (b) a secondantibody, or antigen-binding portion thereof, which specifically bindsto the amino acid sequence of SEQ ID NO: 574 in the lipid binding domainof HCV core protein; and (c) instructions for detecting an HCV coreprotein in a sample.
 24. The kit of claim 23, wherein the first and/orsecond antibody, or the antigen-binding portion thereof, comprises adetectable label attached thereto.